Uses of cerberus, coco and derivatives thereof

ABSTRACT

The disclosure relates to Cerberus/Coco polypeptides or variants thereof for use in treating a variety of disorders associated with myostatin, nodal and GDF-11.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of the filing date of U.S.Application No. 60/873,933, filed Dec. 8, 2006, which is incorporated byreference in its entirety.

BACKGROUND OF THE INVENTION

Transforming growth factor-β superfamily proteins represent a largefamily of cytokines that includes the TGF-βs, activins, inhibins, bonemorphogenetic proteins (BMPs) and Mullerian-inhibiting substance (MIS)(for review, see Massague et al., Trends Cell Biol. 7:187-192, 1997).These proteins contain a conserved C-terminal cysteine-knot motif, andserve as ligands for diverse families of plasma membrane receptors.Members of the TGF-β family exert a wide range of biological effects ona large variety of cell types. Many members of this family haveimportant functions during embryonal development in pattern formationand tissue specification; in the adult, these factors are involved inprocesses such as tissue repair and modulation of the immune system.

Activities of the TGF-β superfamily proteins are regulated throughvarious means. One of the negative regulations for the BMP subfamily ofproteins is through a relatively large family of so-called BoneMorphogenetic Protein (BMP) antagonists/repressors. These BMP repressorsrepresent a subgroup of proteins that bind BMPs, and interfere with BMPbinding to their membrane receptors, thereby antagonizing their actionsduring development and morphogenesis.

The BMP repressors can be further divided into three groups of proteinsbased on structural analysis, especially the number of structurallyconserved Cys residues in their C-terminal characteristic “Cys-knot”structures: the 8-, 9-, or 10-member ring Cys-knot BMP repressors. The8-member ring (CAN subfamily) repressors can be divided further intofour subgroups based on a conserved arrangement of additional cysteineresidues—gremlin and PRDC, Cerberus and coco, and DAN, together withUSAG-1 and sclerostin. Orthologs of these human BMP antagonists in thegenomes of several model organisms have also been identified, and theirphylogenetic relationship has been analyzed (Avsian-Kretchmer and Hsueh,Mol Endocrinol. 18(1): 1-12, 2004, incorporated herein by reference).

Myostatin, or growth/differentiation factor 8 (GDF-8), also belongs tothe transforming growth factor-β (TGF-β) superfamily (McPherron et al.,Nature 387:83-90 (1997)). The human myostatin gene has been cloned(Nestor et al. Proc. Natl. Acad. Sci. 95:14938-43 (1998)), and it hasbeen reported that myostatin immunoreactivity is detectable in humanskeletal muscle in both type 1 and type 2 fibers. With respect tofunction, myostatin may play a role in negatively regulating the growthand development of skeletal muscle (Nestor et al., supra).

A study with myostatin knock-out mice provided the first evidence thatmyostatin is a key negative regulator of muscle development (McPherronet al., Nature 387:83-90 (1997)). In the myostatin null mice, theanimals were significantly larger than wild-type mice and had a largeand widespread increase in skeletal muscle mass. Furthermore, two breedsof cattle, characterized by increased muscle mass, have mutations in themyostatin coding sequence (McPherron et al., Proc. Natl. Acad. Sci.94:12457-61 (1997)). A naturally occurring myostatin reduced-functionmutation in a human child is associated with gross muscle hypertrophyand a family history of exceptional strength (Schuelke et al. 2004 Jun.24; 350(26):2682-8). An antibody against myostatin is reported to havebeneficial effects in animal models of muscle disorders, includingamyotrophic lateral sclerosis (Holzbauer et al. Neurobiol Dis. 2006September; 23(3):697-707).

Additionally, it should be noted that the serum and intramuscularconcentrations of immunoreactive myostatin are increased in HIV-infectedmen with muscle wasting compared with healthy men, and correlateinversely with the fat-free mass index. These data support thehypothesis that myostatin is a negative regulator of skeletal musclegrowth in adult men and contributes to muscle wasting in HIV-infectedmen (Nestor et al., supra).

In view of the above findings, a need exists for a manner of regulatingmyostatin activity, particularly in individuals who experience musclewasting as a result of a condition or disease state such as, forexample, aging related frailty, cachexia in Autoimmune DeficiencySyndrome (AIDS), Multiple Sclerosis, muscular dystrophy, ALS andcancer-cachexia. The present invention provides methods and compositionswhich may be utilized to help individuals with such muscle wastingconditions and provides further insight into the regulation of myostatingene expression.

SUMMARY OF THE INVENTION

In part, the disclosure relates to the discovery that two humanproteins, Cerberus and Coco, that belong to a group of GDF/BMPantagonists, bind to and antagonize myostatin, GDF11 and Nodal, andfurthermore, that the myostatin/GDF11 binding domain resides in thecysteine knot domain of these proteins. Furthermore, with respect toCerberus, myostatin/GDF11 binding and antagonist activity can beseparated from the BMP4/2 binding and antagonist activity. Therefore,the disclosure provides, in part, methods for antagonizing myostatin andGDF11 in vivo by administering polypeptides comprising a myostatinbinding portion of Cerberus or Coco, or variants thereof. One aspect ofthe invention provides polypeptides, and pharmaceutical preparationsthereof, of Cerberus, Coco (from human or non-human animals) or aderivative thereof (collectively herein “Cerberus/Coco proteins”) forinhibiting the function/signaling of Nodal, myostatin, GDF11 and, incertain forms, BMP4 and/or BMP2. In certain embodiments, preparations ofthe subject Cerberus/Coco polypeptides may include variant Cerberus orCoco proteins that retain all or a substantial portion of the bindingaffinity of the parent protein to Nodal, myostatin, GDF11 and/or anotherBMP (such as BMP4). In certain embodiments, preparations of the subjectCerberus/Coco polypeptides include variant Cerberus or Coco proteinsthat retain all or a substantial portion of the binding affinity of theparent protein to myostatin and/or GDF11 while eliminating or reducingbinding to BMP4 and/or BMP2. In certain embodiments, the disclosureprovides the observation that full-length human Cerberus is unstable inthe presence of human serum, and thus altered forms of Cerberus (bothBMP4 binding forms and selective myostatin/GDF11/Nodal binding forms)may be prepared that are stable in the serum for a period of at least 24hours, and optionally 2, 3, 5, 7, 14 or 21 days or longer. Thisobservation may be extrapolated to Coco, and thus altered forms of Cocomay be prepared that are stable in the serum for a period of at least 24hours, and optionally 2, 3, 5, 7, 14 or 21 days or longer. In certainembodiments, the disclosure provides pharmaceutical preparations forinhibiting myostatin, comprising a myostatin antagonist protein thatincludes (at least) a myostatin binding domain of a Cerberus/Cocopolypeptide or variant thereof. The myostatin antagonist protein bindsto and neutralizes one or more of nodal and/or myostatin. Preferably,the pharmaceutical preparation is substantially free of pyrogenicmaterials so as to be suitable for administration as a human orveterinarian therapeutic.

Myostatin is widely recognized as an antagonist of muscle growth.Furthermore, myostatin null mice have shown resistance to obesity anddiabetes under certain conditions. Therefore, the Cerberus/Coco proteinsand pharmaceutical preparations described herein can be used to reducethe severity of a pathologic condition, which is characterized, at leastin part, by an abnormal amount, development or metabolic activity ofmuscle or adipose tissue in a subject. For instance, the pharmaceuticalpreparations of the present invention can be administered in an amounteffective to prevent, ameliorate or reduce the severity of a wastingdisorder, such as age-related wasting, age-related frailty, cachexia,anorexia, Duchenne Muscular Dystrophy (DMD) syndrome, Becker's MuscularDystrophy (BMD) syndrome, facio-scapular-humeral (FSH) musculardystrophy, other muscular dystrophies, AIDS wasting syndrome,neuromuscular diseases, motor neuron diseases, diseases of theneuromuscular junction, and inflammatory myopathies. Excessive BMP4activity has been associated with pathological ossification of variousconnective tissues. Therefore, the Cerberus/Coco proteins andpharmaceutical preparations that retain anti-BMP4 activity can be usedto reduce the severity of a pathologic condition, which ischaracterized, at least in part, by an abnormal ossification in tissuessuch as muscles, tendons, and ligaments. BMP4 is also associated withOsteoarthritis (OA), including the development of osteophytes andsynovial thickening; Fibrodysplasia ossificans progressiva (FOP); andatherosclerosis, especially inflammatory response in early steps ofatherogenesis in lesion-prone areas; and craniosynostoses. Nodalsignaling has been associated with certain cancers, particularlymelanoma. Accordingly, Cerberus/Coco proteins and pharmaceuticalpreparations that retain anti-Nodal activity can be used to treattumors, particularly tumors such as melanomas in which Nodalparticipates in tumor growth and development.

Another aspect of the invention provides a pharmaceutical preparation ofCerberus/Coco protein derivative for specifically inhibiting Nodaland/or myostatin function without substantially compromising BMP (suchas BMP-4) signaling (e.g., does not substantially bind BMP-4 or otherBMPs). Exemplary preparations of this aspect of the invention includepolypeptides including the N-terminal truncated versions of Cerberus orCoco, or other fragments that include the cysteine-core. These so-called“N-terminally truncated Cerberus/Coco derivatives” can be used to reducethe severity of a pathologic condition, which is characterized, at leastin part, by an abnormal amount, development or metabolic activity ofmuscle or adipose tissue in a subject. For instance, the pharmaceuticalpreparations of the present invention can be administered in an amounteffective to prevent, ameliorate or reduce the severity of a wastingdisorder, such as cachexia, anorexia, DMD syndrome, BMD syndrome, AIDSwasting syndrome, muscular dystrophies, neuromuscular diseases, motorneuron diseases, diseases of the neuromuscular junction, andinflammatory myopathies.

In certain embodiments, the mysotatin inhibitor is a polypeptide thatincludes a myostatin binding domain of a Cerberus/Coco protein. Forinstance, the Cerberus protein variant can be derived from a human,mouse, or other species of Cerberus, including a human or mouse Cerberusvariant sequence sharing at least about 50%, 60%, 70%, 80%, 90%, 95%, or99% or more sequence similarity or identity with the human or mouseCerberus protein, and substantially retain the binding affinity ofwild-type Cerberus for myostatin. Likewise, the Coco protein variant canbe derived from a human, mouse, or other species of Coco, including ahuman or mouse Coco variant sequence sharing at least about 50%, 60%,70%, 80%, 90%, 95%, or 99% or more sequence similarity or identity withthe human or mouse Coco protein, and substantially retain the bindingaffinity of wild-type Coco for myostatin.

In certain related embodiments, the mysotatin inhibitor is a polypeptidethat includes a myostatin binding domain of a Cerberus/Coco protein,which polypeptide does not substantially bind BMP-4 or BMP-2. Forinstance, the myostatin binding domain can be derived from a human,mouse, or other species of N-terminally truncated Cerberus, including ahuman or mouse Cerberus derivative, with amino acid residues startingfrom any one of residues 106-119 of SEQ ID No. 1 or 2, and ending at anyresidue after residue 241 of SEQ ID No. 1 or 2, preferably ending at anyresidue between residues 241 and 267 of SEQ ID No. 1 or 2 (all residuenumbers inclusive).

For example, residues 106-119 of human Cerberus are:

PPGTQSLIQPIDGM (SEQ ID NO: 7)

Residues 241-267 of human Cerberus are:

CKVKTEHEDGHILHAGSQDSFIPGVSA (SEQ ID NO: 8)

Also included are Cerberus derived variant sequences, e.g., anN-terminally truncated myostatin binding domain of Cerberus that retainsmyostatin binding activity but loses other BMP binding activity. Variantsequences may be desirable as a way to alter selectivity of theinhibitor (e.g., relative to GDF-8, 11 or nodal binding), alter otherbinding characteristics with respect to myostatin (such as K_(d), and/orK_(on) or K_(off) rates), or improve biodistribution or half life invivo or on the shelf.

In certain preferred embodiments, the Cerberus polypeptide (full-lengthor N-terminally truncated) comprising the myostatin binding domain bindsmyostatin with a K_(d) of 1 μM or less, and more preferably a K_(d) of100 nM, 10 nM or even 1 nM or less.

In certain related embodiments, the mysotatin inhibitor is a polypeptidethat includes a myostatin binding domain of a Coco protein, such as thehuman Coco protein shown in SEQ ID NO:5 or in GenBank Accession number22749329.

In certain preferred embodiments, the Coco polypeptide (full-length orN-terminally truncated) comprising the myostatin binding domain bindsmyostatin with a K_(d) of 1 μM or less, and more preferably a K_(d) of100 nM, 10 nM or even 1 nM or less.

In certain embodiments, the Cerberus/Coco polypeptide (e.g., a myostatinbinding domain thereof) is part of a fusion protein including, inaddition to the myostatin binding domain, one or more polypeptideportions that enhance one or more of in vivo stability, in vivo halflife, uptake/administration, tissue localization or distribution,formation of protein complexes, and/or purification. For instance, thefusion protein can include an immunoglobulin Fc domain. The fusionprotein may include a purification subsequence, such as an epitope tag,a FLAG tag, a polyhistidine sequence, or as a GST fusion.

In certain embodiments, the Cerberus/Coco polypeptide (e.g., myostatinbinding domain thereof) is part of a protein that includes one or moremodified amino acid residues, such as a glycosylated amino acid, aPEGylated amino acid, a farnesylated amino acid, an acetylated aminoacid, a biotinylated amino acid, an amino acid conjugated to a lipidmoiety, or an amino acid conjugated to an organic derivatizing agent.

In certain embodiments, a subject variant Cerberus/Coco polypeptide isselective for binding and inhibition of myostatin, e.g., relative to 11and/or nodal. For instance, the variant Cerberus/Coco polypeptide can beone which has a dissociation constant (K_(d)) for myostatin binding thatis at least 2 times less than its K_(d) for binding 11 and/or nodal, andeven more preferably at least 5, 10, 100 or even 1000 times less.Whether by virtue of binding kinetics or biodistribution, the subjectvariant Cerberus/Coco polypeptide can also be selected based on relativein vivo potency, such as an inhibitor that has an EC₅₀ for inhibitingmyostatin activity, or a particular physiological consequence (such aspromoting muscle growth) that is at least 2 times less than its EC₅₀ forinhibiting 11 and/or nodal activities, and even more preferably at least5, 10, 100 or even 1000 times less.

In certain embodiments, the subject variant Cerberus/Coco polypeptide isselective for binding and inhibition of myostatin, e.g., relative toother BMP proteins such as BMP4. For instance, the variant Cerberus/Cocopolypeptide can be one which has a dissociation constant (K_(d)) formyostatin binding that is at least 2 times less than its K_(d) forbinding BMP4, and even more preferably at least 5, 10, 100 or even 1000times less. Whether by virtue of binding kinetics or biodistribution,the subject variant Cerberus/Coco polypeptide can also be selected basedon relative in vivo potency, such as an inhibitor that has an EC₅₀ forinhibiting myostatin activity, or a particular physiological consequence(such as promoting muscle growth) that is at least 2 times less than itsEC₅₀ for inhibiting BMP4 activities, and even more preferably at least5, 10, 100 or even 1000 times less.

In certain preferred embodiments, the variant Cerberus/Coco polypeptidebinding domain binds myostatin with a K_(d) of 1 μM or less, and morepreferably a K_(d) of 100 nM, 10 nM or even 1 nM or less.

In general, the subject myostatin inhibitor preparations are suitablefor use in a human patients. In preferred embodiments, the subjectpreparations of variant Cerberus/Coco polypeptides will be substantiallyfree of pyrogenic materials so as to be suitable for administration to ahuman patient.

In other embodiments, the subject variant Cerberus/Coco polypeptides canbe administered to non-human animals, particularly other mammals. Forexample, the compounds of the present disclosure can be given tochickens, turkeys, livestock animals (such as sheep, pigs, horses,cattle, etc.), companion animals (e.g., cats and dogs) or may haveutility in aquaculture to accelerate growth and improve the protein/fatratio. To further illustrate, the subject variant Cerberus polypeptidescan be used to stimulate growth or enhance feed efficiency of animalsraised for meat production to improve carcass quality, or to increasemilk production in dairy cattle.

Another aspect of the disclosure relates to packaged pharmaceuticalscomprising a pharmaceutical preparation of a variant Cerberus/Cocopolypeptide, as described herein, and a label or instructions for use inpromoting growth of muscle tissue in a human patient.

Still another aspect of the disclosure relates to packagedpharmaceuticals comprising a pharmaceutical preparation of a variantCerberus/Coco polypeptide, as described herein, and a label orinstructions for veterinarian use in promoting growth of muscle tissuein a non-human mammal.

Another aspect of the disclosure relates to a method for inhibitingmyostatin signal transduction in vivo by administering a pharmaceuticalpreparation of one or more of the subject variant Cerberus/Cocopolypeptides. The subject method can be used to promote muscle growth,promote adipogenic differentiation, and/or promote bone growth ormineralization in human patients or in non-human animals.

In certain embodiments, the treatment methods of the present disclosurecan be used to reduce the severity of a pathologic condition, which ischaracterized, at least in part, by an abnormal amount, development ormetabolic activity of muscle or adipose tissue in a subject. Forinstance, the pharmaceutical preparations of the present disclosure canbe administered in an amount effective to prevent, ameliorate or reducethe severity of a wasting disorder, such as cachexia, anorexia, DMDsyndrome, BMD syndrome, AIDS wasting syndrome, muscular dystrophies,neuromuscular diseases, motor neuron diseases, diseases of theneuromuscular junction, and inflammatory myopathies.

Exemplary muscular dystrophies that can be treated with a regimenincluding the subject myostatin include: Duchenne Muscular Dystrophy(DMD), Becker Muscular Dystrophy (BMD), Emery-Dreifuss MuscularDystrophy (EDMD), Limb-Girdle Muscular Dystrophy (LGMD),Facioscapulohumeral Muscular Dystrophy (FSH or FSHD) (Also known asLandouzy-Dejerine), Myotonic Dystrophy (MMD) (Also known as Steinert'sDisease), Oculopharyngeal Muscular Dystrophy (OPMD), Distal MuscularDystrophy (DD), and Congenital Muscular Dystrophy (CMD).

Exemplary motor neuron diseases that can be treated with a regimenincluding the subject myostatin include: Amyotrophic Lateral Sclerosis(ALS) (Also known as Lou Gehrig's Disease), Infantile Progressive SpinalMuscular Atrophy (SMA, SMA1 or WH) (Also known as SMA Type 1,Werdnig-Hoffman), Intermediate Spinal Muscular Atrophy (SMA or SMA2)(Also known as SMA Type 2), Juvenile Spinal Muscular Atrophy (SMA, SMA3or KW) (Also known as SMA Type 3, Kugelberg-Welander), Spinal BulbarMuscular Atrophy (SBMA) (Also known as Kennedy's Disease and X-LinkedSBMA), and Adult Spinal Muscular Atrophy (SMA).

Exemplary inflammatory myopathies that can be treated with a regimenincluding the subject myostatin include: Dermatomyositis (PM/DM),Polymyositis (PM/DM), and Inclusion Body Myositis (IBM).

Exemplary diseases of the neuromuscular junction that can be treatedwith a regimen including the subject myostatin include: MyastheniaGravis (MG), Lambert-Eaton Syndrome (LES), and Congenital MyasthenicSyndrome (CMS).

Exemplary myopathies due to endocrine abnormalities that can be treatedwith a regimen including the subject myostatin include: HyperthyroidMyopathy (HYPTM) and Hypothyroid Myopathy (HYPOTM).

Exemplary diseases of peripheral nerve that can be treated with aregimen including the subject myostatin include: Charcot-Marie-ToothDisease (CMT), Dejerine-Sottas Disease (DS), and Friedreich's Ataxia(FA).

Other exemplary myopathies that can be treated with a regimen includingthe subject myostatin include: Myotonia Congenita (MC), ParamyotoniaCongenita (PC), Central Core Disease (CCD), Nemaline Myopathy (NM),Myotubular Myopathy (MTM or MM), and Periodic Paralysis (PP).

Exemplary metabolic diseases of muscle that can be treated with aregimen including the subject myostatin include: PhosphorylaseDeficiency (MPD or PYGM), Acid Maltase Deficiency (AMD),Phosphofructokinase Deficiency (PFKM), Debrancher Enzyme Deficiency(DBD), Mitochondrial Myopathy (MITO), Carnitine Deficiency (CD),Carnitine Palmityl Transferase Deficiency (CPT), Phosphoglycerate KinaseDeficiency (PGK), Phosphoglycerate Mutase Deficiency (PGAM or PGAMM),Lactate Dehydrogenase Deficiency (LDHA), and Myoadenylate DeaminaseDeficiency (MAD).

The subject method can also be used to prevent, ameliorate or reduce theseverity of a metabolic disorder, such as in the treatment of obesity ortype II diabetes. To further illustrate, the subject variantCerberus/Coco polypeptide preparations can be used to decrease body fatproportion in a subject.

In still other embodiments, the variant Cerberus/Coco polypeptidepreparations can be used as part of such methods as reducing frailtyassociated with aging.

The subject pharmaceutical composition can also be used as myostatinantagonist to treat a number of neuronal system disease conditions,including CNS injuries/disease such as spinal cord injury and stroke,and PNS injuries/diseases.

In one aspect, the disclosure provides a myostatin antagonist proteincomprising an amino acid sequence that is at least 90% identical to thesequence of amino acids 162-241 of human Cerberus (SEQ ID NO:2), andwherein said protein is substantially serum stable for a period of atleast 24 hours.

In certain embodiments, the myostatin antagonist protein comprises anamino acid sequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or100% identical to one or more of the following: the sequence of aminoacids 156-241 of human Cerberus, the sequence of amino acids 156-267 ofhuman Cerberus, the sequence of amino acids 141-241 of human Cerberus,the sequence of amino acids 141-267 of human Cerberus, the sequence ofamino acids 119-241 of human Cerberus, the sequence of amino acids41-241 of human Cerberus, the sequence of amino acids 41-267 of humanCerberus, the sequence of amino acids 18-241 of human Cerberus, or thesequence of amino acids 18-267 of human Cerberus.

In certain embodiments, the myostatin antagonist protein retains atleast 50% of the myostatin antagonist activity after exposure to humanserum for 24 hours at 37° C. The myostatin antagonist activity may beassessed, for example, in an A204 cell based assay.

In certain embodiments, the myostatin antagonist protein comprises amodification with respect to the amino acid sequence of SEQ ID NO:2 suchthat cleavage in human serum is reduced or eliminated. The modificationwith respect to the amino acid sequence of SEQ ID NO:2 may reduce oreliminate cleavage within one or more of the following sequences: thesequence SHCLPAK (SEQ ID NO: 22) of human Cerberus, the sequence MFRKTP(SEQ ID NO: 23) of human Cerberus, or the sequence NQRELP (SEQ ID NO:24) of human Cerberus.

In another aspect, the disclosure provides a myostatin antagonistprotein, the protein comprising an amino acid sequence that is at least90% identical to the sequence of amino acids 101-185 of human Coco (SEQID NO:5), and wherein said protein is substantially serum stable for aperiod of at least 24 hours.

In certain embodiments, the myostatin antagonist protein comprises amodification with respect to the amino acid sequence of SEQ ID NO:5 suchthat cleavage in human serum is reduced or eliminated. The modificationwith respect to the amino acid sequence of SEQ ID NO:5 may reduce oreliminate cleavage within one or both of the following sequences: PARKRW(SEQ ID NO: 25) or SRRRVK (SEQ ID NO: 26) of human Coco.

In certain embodiments, the myostatin antagonist protein retains atleast 50% of the myostatin antagonist activity after exposure to humanserum for 24 hours at 37° C. The myostatin antagonist activity may beassessed, for example, in an A204 cell based assay.

In certain embodiments, the myostatin antagonist protein comprises amodification with respect to the amino acid sequence of SEQ ID NO:5 suchthat cleavage in human serum is reduced or eliminated. The modificationwith respect to the amino acid sequence of SEQ ID NO:5 may reduce oreliminate cleavage within one or both of the following sequences: PARKRWor SRRRVK of human Coco.

In certain embodiments, the myostatin antagonist protein may be a fusionprotein including one additional polypeptide portion that enhances oneor more of in vivo stability, in vivo half life, uptake/administration,tissue localization or distribution, formation of protein complexes,and/or purification. In certain embodiments, the fusion protein includesa portion of an immunoglobulin heavy chain constant domain. In certainembodiments, the fusion protein comprises an Fc domain of animmunoglobulin. In certain embodiments, the myostatin antagonist proteinincludes one or more modified amino acid residues selected from: aglycosylated amino acid, a PEGylated amino acid, a farnesylated aminoacid, an acetylated amino acid, a biotinylated amino acid, an amino acidconjugated to a lipid moiety, and an amino acid conjugated to an organicderivatizing agent.

In certain embodiments, the myostatin antagonist protein is a fusionprotein that further comprises a second myostatin inhibitor domain,which is a polypeptide affinity reagent that selectively binds tomyostatin and competes with the binding of an ALK7 or ALK4 receptor. Incertain embodiments, the affinity reagent is one or more of thefollowing: (i) an antibody agent, (ii) a peptide or scaffolded peptidethat selectively binds to myostatin and competes with the binding of anALK7 or ALK4 receptor, (iii) a myostatin binding domain of ALK7 or ALK4,or (iv) small organic molecule that selectively binds to myostatin andcompetes with the binding of an ALK7 or ALK4 receptor. Examples ofsuitable antibody agents include, for example, a recombinant antibody; amonoclonal antibody; a V_(H) domain; a V_(L) domain; an scFv; an Fabfragment; an Fab′ fragment; an F(ab′)₂; an Fv; or a disulfide linked Fv.In certain embodiments, the antibody agent is a fully human antibody ora humanized chimeric antibody, or an antigen binding fragment thereof.

In another aspect, the disclosure provides a pharmaceutical preparationcomprising one or more of the myostatin antagonist proteins describedherein.

In another aspect, the disclosure provides a method for inhibitingmyostatin and/or GDF11 and/or Nodal in a patient, the method comprisingadministering to the patient an effective amount of one or more of themyostatin antagonist proteins described herein. In certain embodiments,inhibiting myostatin and/or GDF11 and/or Nodal in a patient causes adetectable change in the expression of a gene that is regulated bymyostatin and/or GDF11 and/or Nodal.

In another aspect, the disclosure provides a method for increasingmuscle mass in a patient, the method comprising administering to thepatient an effective amount of one or more of the myostatin antagonistproteins described herein.

In another aspect, the disclosure provides a pharmaceutical preparationsubstantially free of pyrogenic materials, comprising a myostatinantagonist protein including a myostatin binding domain of a Cerberus orCoco polypeptide or variant thereof, which myostatin antagonist protein:(a) binds to and inhibits the signaling activity of one or more ofNodal, GDF11 and/or myostatin; and (b) does not substantially bind toBMP4.

In certain embodiments, the myostatin antagonist protein promotes growthof muscle tissue.

In certain embodiments, the myostatin binding domain has an amino acidsequence that is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100%identical to one or both of the following: amino acids 162-241 of SEQ IDNO: 2, amino acids 101-189 of SEQ ID NO:5. In certain embodiments, themyostatin binding domain has an amino acid sequence that is at identicalto an amino acid sequence selected from the group consisting of: aminoacids 162-241 of SEQ ID NO: 2 and amino acids 101-189 of SEQ ID NO:5, orany naturally occurring human allelic variant thereof.

In certain embodiments, the myostatin antagonist protein does notinclude a full-length mature human Cerberus protein.

In certain embodiments, the myostatin antagonist protein is a fusionprotein including one additional polypeptide portion that enhance one ormore of in vivo stability, in vivo half life, uptake/administration,tissue localization or distribution, formation of protein complexes,and/or purification. In certain embodiments, the fusion protein includesa portion of an immunoglobulin heavy chain constant domain. In certainembodiments, the fusion protein comprises an Fc domain of animmunoglobulin.

In certain embodiments, the myostatin antagonist protein includes one ormore modified amino acid residues selected from: a glycosylated aminoacid, a PEGylated amino acid, a farnesylated amino acid, an acetylatedamino acid, a biotinylated amino acid, an amino acid conjugated to alipid moiety, and an amino acid conjugated to an organic derivatizingagent.

In certain embodiments, the myostatin antagonist protein is a fusionprotein that further comprises a second myostatin inhibitor domain,which is a polypeptide affinity reagent that selectively binds tomyostatin and competes with the binding of an ALK7 or ALK4 receptor. Incertain embodiments, the affinity reagent is one or more of thefollowing: (i) an antibody agent, (ii) a peptide or scaffolded peptidethat selectively binds to myostatin and competes with the binding of anALK7 or ALK4 receptor, (iii) a myostatin binding domain of ALK7 or ALK4,or (iv) a small organic molecule that selectively binds to myostatin andcompetes with the binding of an ALK7 or ALK4 receptor. Examples ofsuitable antibody agents include, for example, a recombinant antibody; amonoclonal antibody; a V_(H) domain; a V_(L) domain; an scFv; an Fabfragment; an Fab′ fragment; an F(ab′)₂; an Fv; or a disulfide linked Fv.In certain embodiments, the antibody agent is a fully human antibody ora humanized chimeric antibody, or an antigen binding fragment thereof.

In another aspect, the disclosure provides a method for inhibitingmyostatin and/or GDF11 in a patient, the method comprising administeringto the patient an effective amount of a myostatin antagonist proteinincluding a myostatin binding domain of a Cerberus or Coco polypeptideor variant thereof, which myostatin antagonist protein binds to andinhibits the signaling activity of one or more of nodal, GDF11 and/ormyostatin. In certain embodiments, the myostatin binding domain has anamino acid sequence that is at least 90% identical to an amino acidsequence selected from the group consisting of: amino acids 162-241 ofSEQ ID NO: 2 and amino acids 101-189 of SEQ ID NO:5. In certainembodiments, the myostatin binding domain has an amino acid sequencethat is at identical to an amino acid sequence selected from the groupconsisting of: amino acids 162-241 of SEQ ID NO: 2 and amino acids101-189 of SEQ ID NO:5, or any naturally occurring human allelic variantthereof. In certain embodiments, inhibiting myostatin and/or GDF11 in apatient causes a detectable change in the expression of a gene that isregulated by myostatin and/or GDF11. In certain embodiments, themyostatin antagonist does not substantially bind to BMP4.

In another aspect, the disclosure provides a method for increasingskeletal muscle mass in a patient in need thereof, the method comprisingadministering to the patient an effective amount of a myostatinantagonist protein including a myostatin binding domain of a Cerberus orCoco polypeptide or variant thereof, which myostatin antagonist proteinbinds to and inhibits the signaling activity of one or more of nodal,GDF11 and/or myostatin. In certain embodiments, the myostatin bindingdomain has an amino acid sequence that is at least 80% identical to anamino acid sequence selected from the group consisting of: amino acids162-241 of SEQ ID NO: 2 and amino acids 101-189 of SEQ ID NO:5. Incertain embodiments, the myostatin binding domain has an amino acidsequence that is at identical to an amino acid sequence selected fromthe group consisting of: amino acids 162-241 of SEQ ID NO: 2 and aminoacids 101-189 of SEQ ID NO:5, or any naturally occurring human allelicvariant thereof. In certain embodiments, the myostatin antagonist doesnot substantially bind to BMP4.

In another aspect, the disclosure provides use of a myostatin antagonistprotein including a myostatin antagonist protein including a myostatinbinding domain of a Cerberus or Coco polypeptide or variant thereof,which myostatin antagonist protein binds to and inhibits the signalingactivity of one or more of nodal, GDF11 and/or myostatin for thepreparation of a medicament for promoting growth of muscle tissue in amammal.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a schematic drawing of where Wnt, Nodal and BMP bind toCerberus. BMP-2 and the highly related BMP4 competitively bind Cerberus,likely in the same region. Other more distantly related or unrelatedproteins, such as TGF-beta1, EGF, and PDGF, do not compete with BMP-4.The N-terminally truncated version of Cerberus still binds Xnr-1(Xenopus homolog of mouse Nodal). (Adapted from Piccolo et al., Nature397: 707-710, 1999).

FIG. 2 Binding of Caronte-Fc to 11. The tracing shows that Caronte-Fcbinds to 11 on a BiaCore chip. 11 was immobilized on a BiaCore CM5 chipusing standard amine coupling procedure. Trace: Caronte-Fc (200 μg/ml;R&D Systems) was injected on the 11 coupled chip.

FIG. 3 A-204 Reporter Gene Assay. The figure shows the Reporter vector:pGL3(CAGA)12 (described in Dennler et al, 1998, EMBO 17: 3091-3100.) TheCAGA12 motif (SEQ ID NO: 30) is present in TGF-Beta responsive genes(PAI-1 gene), so this vector is of general use for factors signalingthrough Smad2 and 3.

FIG. 4 Caronte-Fc inhibits 11 signaling in the A-204 Reporter GeneAssay. An ActRIIA-Fc (“IIA muG2a”) fusion also inhibits 11 signaling.

FIG. 5 Caronte-Fc does not inhibit Activin A in the A-204 Reporter GeneAssay. An ActRIIA-Fc fusion (“IIA muG2a”), as expected, does inhibitActivin A signaling.

FIG. 6 Cerberus-Fc and Caronte-Fc both inhibit GDF-8 signaling in theA-204 Reporter Gene Assay.

FIG. 7 Human Cerberus-Fc is degraded in human serum. Conditioned mediumfrom cells expressing human Cerberus-Fc was incubated overnight at 37deg. C. with varying amounts of human serum (percentages of serum addedare shown at top), and resolved by SDS-PAGE. Cerberus was detected byWestern blot with a primary antibody: biotinylated polyclonalanti-cerberus, and a secondary antibody: avidin-HRP. The left lane ismolecular weight standards. The major band, at roughly 70 kD isCerberus-Fc, which is completely degraded when incubated with 5% humanserum.

FIG. 8 Human Coco-Fc (murine Fc) inhibits 11 signaling in a cell basedassay. Conditioned medium from cells expressing human Coco-mFc wastested for effects on A-204 reporter gene expression in the presence of11.

DETAILED DESCRIPTION I. Overview

Cerberus is expressed in the anterior endomesoderm (Bouwmeester et al.,Nature 382: 595-601, 1996; Piccolo et al., Nature 397: 707-10, 1999;Rodriguez et al., Nature 401: 243-51, 1999) during development. Caronte,a chick ortholog, is involved in left-right asymmetry in the chickembryo (Rodriguez, supra). Cerberus functions as a multivalent growthfactor antagonist in the extracellular space and inhibits signaling byBMP-4, nodal, and Wnt (Belo et al., Genesis 26: 265-70, 2000). MouseCerberus binds to BMP proteins and nodal via independent sites (Piccolo,supra), whereas the Xenopus Cerberus also binds Wnt proteins andinhibits their actions (Belo, supra). Cerberus has the unique propertyof inducing ectopic heads in the absence of trunk structures (Piccolo,supra). The expression of Cerberus during gastrulation is activated bynodal-related signals in endoderm and by Spemann-organizer factors(Yamamoto et al., Dev Biol 257: 190-204, 2003).

Orthologs for Cerberus can be found in Xenopus tropicalis and Fugurubripes, but are missing in invertebrates. In Fugu rubripes, there isonly one ortholog for Cerberus. All orthologous genes for Cerberus havetwo exons; the first eight amino acids of the cystine-knot domain areencoded by the 3′ end of the first exon and the remainder of the motifby the second exon. In some orthologs, a predicted proteolytic cleavagesite can be found upstream of the beginning of the cystine-knot domain.

Coco is another member of the Cerberus/Dan family of proteins thatinhibits Nodal signaling.

In part, the present disclosure provides Coco or Cerberus derivativesfor inhibiting Nodal, 11 and/or myostatin function. In certainembodiments, the Coco and Cerberus derivatives inhibit Nodal, 11 and/ormyostatin function without substantially compromising BMP (such asBMP-4) signaling (e.g., does not substantially bind BMP-4 or otherBMPs). The subject Cerberus derivatives may also be used to inhibit BMP(such as BMP-4) signaling.

Exemplary preparations of the subject disclosure include Cerberuspolypeptide derivatives, including the N-terminal truncated versions ofCerberus or Coco. These so-called “Cerberus derivatives” or “Cocoderivatives” can be used to reduce the severity of a pathologiccondition, which is characterized, at least in part, by an abnormalamount, development or metabolic activity of muscle or adipose tissue ina subject. For instance, the pharmaceutical preparations of the presentdisclosure can be administered in an amount effective to prevent,ameliorate or reduce the severity of a wasting disorder, such ascachexia, anorexia, DMD syndrome, BMD syndrome, AIDS wasting syndrome,muscular dystrophies, neuromuscular diseases, motor neuron diseases,diseases of the neuromuscular junction, and inflammatory myopathies.

II. Definitions

The terms used in this specification generally have their ordinarymeanings in the art, within the context of this disclosure and in thespecific context where each term is used. Certain terms are discussedbelow or elsewhere in the specification, to provide additional guidanceto the practitioner in describing the compositions and methods of thedisclosure and how to make and use them. The scope an meaning of any useof a term will be apparent from the specific context in which the termis used.

“About” and “approximately” shall generally mean an acceptable degree oferror for the quantity measured given the nature or precision of themeasurements. Typically, exemplary degrees of error are within 20percent (%), preferably within 10%, and more preferably within 5% of agiven value or range of values.

Alternatively, and particularly in biological systems, the terms “about”and “approximately” may mean values that are within an order ofmagnitude, preferably within 5-fold and more preferably within 2-fold ofa given value. Numerical quantities given herein are approximate unlessstated otherwise, meaning that the term “about” or “approximately” canbe inferred when not expressly stated.

The methods of the disclosure may include steps of comparing sequencesto each other, including wild-type sequence to one or moremutants/sequence variants Such comparisons typically comprise alignmentsof polymer sequences, e.g., using sequence alignment programs and/oralgorithms that are well known in the art (for example, BLAST, FASTA andMEGALIGN, to name a few). The skilled artisan can readily appreciatethat, in such alignments, where a mutation contains a residue insertionor deletion, the sequence alignment will introduce a “gap” (typicallyrepresented by a dash, or “A”) in the polymer sequence not containingthe inserted or deleted residue.

“Homologous,” in all its grammatical forms and spelling variations,refers to the relationship between two proteins that possess a “commonevolutionary origin,” including proteins from superfamilies in the samespecies of organism, as well as homologous proteins from differentspecies of organism. Such proteins (and their encoding nucleic acids)have sequence homology, as reflected by their sequence similarity,whether in terms of percent identity or by the presence of specificresidues or motifs and conserved positions.

The term “sequence similarity,” in all its grammatical forms, refers tothe degree of identity or correspondence between nucleic acid or aminoacid sequences that may or may not share a common evolutionary origin.

However, in common usage and in the instant application, the term“homologous,” when modified with an adverb such as “highly,” may referto sequence similarity and may or may not relate to a commonevolutionary origin.

A nucleic acid molecule is “hybridizable” to another nucleic acidmolecule, such as a cDNA, genomic DNA, or RNA, when a single strandedform of the nucleic acid molecule can anneal to the other micleic acidmolecule under the appropriate conditions of temperature and solutionionic strength (see Sambrook et al. Molecular Cloning: A LaboratoryManual, Second Edition (1989) Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.). The conditions of temperature and ionic strengthdetermine the “stringency” of the hybridization. For preliminaryscreening for homologous nucleic acids, low stringency hybridizationconditions, corresponding to a T_(m) (melting temperature) of 55° C.,can be used, e.g., 5×SSC, 0.1% SDS, 0.25% milk, and no formamide; or 30%formamide, 5×SSC, 0.5% SDS).

Moderate stringency hybridization conditions correspond to a higherT_(m), e.g., 40% formamide, with 5× or 6×SSC. High stringencyhybridization conditions correspond to the highest T_(m), e.g., 50%formamide, 5× or 6×SSC. SSC is 0.15 M NaCl, 0.015 M Na-citrate.

“High stringency condition” is well understood in the art to encompassconditions of hybridization which allow hybridization of structurallyrelated, but not structurally dissimilar, nucleic acids. The term“stringent” is a term of art which is understood by the skilled artisanto describe any of a number of alternative hybridization and washconditions which allow annealing of only highly complementary nucleicacids.

Exemplary high stringent hybridization conditions is equivalent to about20-27° C. below the melting temperature (T_(m)) of the DNA duplex formedin about 1 M salt. Many equivalent procedures exist and several popularmolecular cloning manuals describe suitable conditions for stringenthybridization and, furthermore, provide formulas for calculating thelength of hybrids expected to be stable under these conditions (see e.g.Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989),6.3.1-6 or 13.3.6; or pages 9.47-9.57 of Sambrook, et al. (1989)Molecular Cloning, 2^(nd) ed., Cold Spring Harbor Press).

Hybridization requires that the two nucleic acids contain complementarysequences, although depending on the stringency of the hybridization,mismatches between bases are possible. The appropriate stringency forhybridizing nucleic acids depends on the length of the nucleic acids andthe degree of complementation, variables well known in the art. Thegreater the degree of similarity or homology between two nucleotidesequences, the greater the value of T_(m), for hybrids of nucleic acidshaving those sequences. The relative stability (corresponding to higherT_(m)) of micleic acid hybridizations decreases in the following order:RNA:RNA, DNA:RNA, DNA:DNA. For hybrids of greater than 100 nucleotidesin length, equations for calculating T_(m) have been derived (seeSambrook et al., supra, 9.51). For hybridization with shorter nucleicacids, i.e., oligonucleotides, the position of mismatches becomes moreimportant, and the length of the oligonucleotide determines itsspecificity (see Sambrook et al., supra, 11.8). A minimum length for ahybridizable nucleic acid is at least about 10 nucleotides; preferablyat least about 15 nucleotides; and more preferably the length is atleast about 20 nucleotides.

Unless specified, the term “standard hybridization conditions” refers toa T_(m) of about 55° C., and utilizes conditions as set forth above. Ina preferred embodiment, the T_(m) is 60° C.; in a more preferredembodiment, the T_(m) is 65° C. In a specific embodiment, “highstringency” refers to hybridization and/or washing conditions at 68° C.in 0.2×SSC, at 42° C. in 50% formamide, 4×SSC, or under conditions thatafford levels of hybridization equivalent to those observed under eitherof these two conditions.

Suitable hybridization conditions for oligonucleotides (e.g., foroligonucleotide probes or primers) are typically somewhat different thanfor full-length nucleic acids (e.g., full-length cDNA), because of theoligonucleotides' lower melting temperature. Because the meltingtemperature of oligonucleotides will depend on the length of theoligonucleotide sequences involved, suitable hybridization temperatureswill vary depending upon the oligonucleotide molecules used. Exemplarytemperatures may be 37° C. (for 14-base oligonucleotides), 48° C. (for17-base oligonucleotides), 55° C. (for 20-base oligonucleotides) and 60°C. (for 23-base oligonucleotides). Exemplary suitable hybridizationconditions for oligonucleotides include washing in 6×SSC, 0.05% sodiumpyrophosphate, or other conditions that afford equivalent levels ofhybridization.

“Polypeptide,” “peptide” or “protein” are used interchangeably todescribe a chain of amino acids that are linked together by chemicalbonds called “peptide bonds.” A protein or polypeptide, including anenzyme, may be a “native” or “wild-type,” meaning that it occurs innature; or it may be a “mutant,” “variant,” or “modified,” meaning thatit has been made, altered, derived, or is in some way different orchanged from a native protein or from another mutant.

As used herein, the term “Cerberus/Coco protein” is used to signify thehuman Cerberus and Coco proteins, as well as homologs from other species(e.g., Caronte is the chicken Cerberus homolog) and derivatives(including forms with altered sequences and truncated forms) that retaina biological activity of the naturally occurring form.

“Cerberus or Cerberus-like protein” refers to mammalian Cerberus andCerberus-like proteins, such as the murine (NCBI RefSeq ID NP_(—)034017)or human (NCBI RefSeq ID NP_(—)005445) Cerberus proteins (also see SEQID Nos. 2 and 8, respectively, of US 2002/0164682 A1, the entirecontents of which is incorporated herein by reference), and otherproteins which share sequence homology to the highly conserved cysteinepattern of the C-terminal portion of the mammalian Cerberus proteins.Exemplary amino acid sequences for Cerberus proteins include

Murine Cerberus protein (NCBI RefSeq ID NP_034017) (SEQ ID NO: 1):   1MHLLLVQLLV LLPLGKADLC VDGCQSQGSL SFPLLERGRR DLHVANHEEA EDKPDLFVAV  61PHLMGTSLAG EGQRQRGKML SRLGRFWKKP ETEFYPPRDV ESDHVSSGMQ AVTQPADGRK 121VERSPLQEEA KRFWERFMFR KGPAFQGVIL PIKSHEVHWE TCRTVPFNQT IAHEDCQKVV 181VQNNLCFGKC SSIRFPGEGA DAHSFCSHCS PTKFTTVHLM LNCTSPTPVV KMVMQVEECQ 241CMVKTERGEE RLLLAGSQGS FIPGLPASKT NP Human Cerberus protein (NCBI RefSeqID NP_005445) (SEQ ID NO: 2):   1 MHLLLFQLLV LLPLGKTTRH QDGRQNQSSLSPVLLPRNQR ELPTGNHEEA EEKPDLFVAV  61 PHLVATSPAG EGQRQREKML SRFGRFWKKPEREMHPSRDS DSEPFPPGTQ SLIQPIDGMK 121 MEKSPLREEA KKFWHHFMFR KTPASQGVILPIKSHEVHWE TCRTVPFSQT ITHEGCEKVV 181 VQNNLCFGKC GSVHFPGAAQ HSHTSCSHCLPAKFTTMHLP LNCTELSSVI KVVMLVEECQ 241 CKVKTEHEDG HILHAGSQDS FIPGVSA

The mouse and human Cerberus are as disclosed in US 2002/0164682 A1, asSEQ ID NOs. 1 and 7 (incorporated herein by reference).

NM_009887.1 (mouse Cerberus mRNA) (SEQ ID NO: 3).    1 ggggggggggggggtcagag ggagctttct tttaggcccg tccatctgtg aatctaacct   61 cagtttctgggaatcaggaa gcatgcatct cctcttagtt cagctgcttg ttctcttgcc  121 tctggggaaggcagacctat gtgtggatgg ctgccagagt cagggctctt tatcctttcc  181 tctcctagaaaggggtcgca gagatctcca cgtggccaac cacgaggagg cagaagacaa  241 gccggatctgtttgtggccg tgccacacct catgggcacc agcctggctg gggaaggcca  301 gaggcagagagggaagatgc tgtccaggct tggaagattc tggaagaaac ctgagaccga  361 attttaccccccaagggatg tggaaagcga tcatgtctca tcggggatgc aggccgtgac  421 tcagccagcagatgggagga aagtggagag atcacctcta caggaggaag ccaagaggtt  481 ctggcatcggttcatgttca gaaagggccc ggcgttccag ggagtcatcc tgcccatcaa  541 aagccacgaagtacactggg agacctgcag gactgtgccc ttcaaccaga ccattgccca  601 tgaagactgtcaaaaagtcg ttgtccagaa caacctttgc tttggcaaat gcagttccat  661 tcgttttcccggagaagggg cagatgccca cagcttctgc tcccactgct cgcccaccaa  721 attcaccaccgtgcacttga tgctgaactg caccagccca acccccgtgg tcaagatggt  781 gatgcaagtagaagagtgtc agtgcatggt gaagacggaa cgtggagagg agcgcctcct  841 actggctggttcccagggtt ccttcatccc tggacttcca gcttcaaaaa caaacccatg  901 aattacctcaacagaaagca aaacctcaac agaataagtg agggttattc aatctggaaa  961 tgttatgtgagttatataaa gatcagtgga aaatatcttt ctctctccct ctctccccct 1021 ctctcttctctctattttct ctctctctct ctctctctct ctctctctct ctctctctca 1081 cacacacacacacacacaca cacacacaca catgtttgtg tttagacagg gtcttatgta 1141 ttctcagctggcctcaaact cacaatgtgg ctggggatga ttttaaactc ctgatccaat 1201 tcctgagtgctgggattaca gacatgctcc ataanacata gctcccagaa ggatttttaa 1261 aagagattttgcatgtttca aagttgcctt tgagactcag aaatattttg atntattgaa 1321 tggccttgccacagatgtgg gaggcagctt gcttggtggc ccaagtattt tttttttgtt 1381 cgttcagaattctccacatg aagtttttac tgttggttat ctggcgttga agaaggaata 1441 gtgaaggtacttttaacagt ttacacgtgg aaggggctca ggcactagga accaaccttt 1501 tcccggaatatgaggaaaat acatgaacag tattagagtc acttgaggaa gttactagga 1561 aacgccataagtctccaagt acattgtgag tcattttgaa ggacaatcgt gtatatagac 1621 gaaatcttctactcgtatgc ttttgaatct tctagcaagt taggtttcta tgtttgggct 1681 tcttcctattgtctaagagt atgtgtgaca aattcaacct gacaaatacc tcaatggcaa 1741 attctgaccctg NCBI RefSeq ID NM_005454.1 (human Cerberus mRNA) (SEQ ID NO: 4).    1atgcatctcc tcttatttca gctgctggta ctcctgcctc taggaaagac cacacggcac   61caggatggcc gccagaatca gagttctctt tcccccgtac tcctgccaag gaatcaaaga  121gagcttccca caggcaacca tgaggaagct gaggagaagc cagatctgtt tgtcgcagtg  181ccacaccttg tagccaccag ccctgcaggg gaaggccaga ggcagagaga gaagatgctg  241tccagatttg gcaggttctg gaagaagcct gagagagaaa tgcatccatc cagggactca  301gatagtgagc ccttcccacc tgggacccag tccctcatcc agccgataga tggaatgaaa  361atggagaaat ctcctcttcg ggaagaagcc aagaaattct ggcaccactt catgttcaga  421aaaactccgg cttctcaggg ggtcatcttg cccatcaaaa gccatgaagt acattgggag  481acctgcagga cagtgccctt cagccagact ataacccacg aaggctgtga aaaagtagtt  541gttcagaaca acctttgctt tgggaaatgc gggtctgttc attttcctgg agccgcgcag  601cactcccata cctcctgctc tcactgtttg cctgccaagt tcaccacgat gcacttgcca  661ctgaactgca ctgaactttc ctccgtgatc aaggtggtga tgctggtgga ggagtgccag  721tgcaaggtga agacggagca tgaagatgga cacatcctac atgctggctc ccaggattcc  781tttatcccag gagtttcagc ttga

It is also expected that Cerberus related proteins also exist in otherspecies, including family members in Xenopus, and Drosophila, C.elegans, zebrafish, as well as in all mammals, for example, rats, miceand humans. “Cerberus or Cerberus-like proteins” also includes variantsof the Cerberus proteins, such as allelic variants or variants inducedby mutagenesis or deletions, and fragments of Cerberus proteins whichvariants and fragments retain myostatin binding activity.“Cerberus-like” proteins is also used to signify the family of proteinssharing structural and/or functional similarity, including thoseproteins which are described further herein. Such proteins may haveamino acid sequences sharing significant sequence identity (e.g., atleast about 50%, 60%, 70%, 80%, 90%, 95%, 99% or more) with the human ormouse Cerberus proteins, over the full-length, or at least within themyostatin binding domain of the human or mouse Cerberus. Cerberus-likeproteins also include proteins that have amino acid sequences that areencoded by nucleic acid sequences that hybridize under stringentconditions with the coding sequences for human or mouse Cerberus,particularly that portion of the coding sequence for the myostatinbinding domain. A Cerberus derivative or variant sequence may or may notlack the N-terminal BMP binding domain. A variety of allelic variants ofhuman Cerberus are known, including A65G (alanine 65 to glycine), V179Iand L221V.

“Coco or Coco-like protein” refers to mammalian Coco proteins andrelated homologs, such as the human Coco protein of GenBank Accession22749329, and other proteins which share sequence homology to the highlyconserved cysteine pattern of the C-terminal portion of the mammalianCoco proteins. An exemplary amino acid sequences for human Coco proteinis

(SEQ ID NO: 5)   1 MLLGQLSTLL CLLSGALPTG SGRPEPQSPR PQSWAAANQTWALGPGALPP LVPASALGSW  61 KAFLGLQKAR QLGMGRLQRG QDEVAAVTLP LNPQEVIQGMCKAVPFVQVF SRPGCSAIRL 121 RNHLCFGNCS SLYIPGSDPT PLVLCNSCMP ARKRWAPVVLWCLTGSSASR RRVKISTMLI 181 EGCHCSPKA

Amino acids 1-21 of SEQ ID NO:5 correspond to a signal peptide that maybe replaced with an alternative leader sequence. A mature secreted Cocopolypeptide is expected to correspond to amino acids 22-189 of SEQ IDNO:5, although imprecisions in the signal peptide processing enzymes maylead to alternative or additional cleavage at positions ranging from oneto five amino acids towards the amino terminus or the carboxy terminusfrom the glycine at position 22. As disclosed herein, a tPA leadersequence or other heterologous leader sequence may be used in place ofthe native leader sequence. Proposed leader sequences are as follows:

 (i) Honey bee mellitin (HBML): MKFLVNVALVFMVVYISYIYA (SEQ ID NO: 14)(ii) Tissue Plasminogen Activator (TPA): MDAMKRGLCCVLLLCGAVFVSP (SEQ IDNO: 15)

The human Coco coding sequence is disclosed in GenBank Accession22749328 (incorporated herein by reference) (SEQ ID NO:6).

   1 agtccggaca gacagacagg cagacagacg cacggacaag cagatgctcc ttggccagct  61 atccactctt ctgtgcctgc ttagcggggc cctgcctaca ggctcaggga ggcctgaacc 121 ccagtctcct cgacctcagt cctgggctgc agccaatcag acctgggctc tgggcccagg 181 ggccctgccc ccactggtgc cagcttctgc ccttgggagc tggaaggcct tcttgggcct 241 gcagaaagcc aggcagctgg ggatgggcag gctgcagcgt gggcaagacg aggtggctgc 301 tgtgactctg ccgctgaacc ctcaggaagt gatccagggg atgtgtaagg ctgtgccctt 361 cgttcaggtg ttctcccggc ccggctgctc agccatacgc ctccgaaatc atctgtgctt 421 tggtcattgc tcctctctct acatccctgg ctcggacccc accccactag tcctgtgcaa 481 cagctgtatg cctgctcgca agcgttgggc acccgtggtc ctgtggtgtc tcactggcag 541 ctcagcctcc cgtcgacggg tgaagatatc caccatgctg atcgaggggt gtcactgcag 601 cccaaaagca tgaactgagc atcgtggatg ggtgcacgga gacacgcacc ttggagaaat 661 gaggggagat ggaccaagaa agacgtggac ctggatgatg tactctgggt caagagacca 721 gggatgcagg gttaggcaga caggtcccca gagtcctcac cctgctcccc agacagtaga 781 cacagtgccc gtcctggagt tgcaccactg atagtcacag cacacaatga ttgacaactc 841 actttttttt ttttttttga gatggagtct cgctctgtcg cccaggctgg agtgcagtgg 901 cgcaatctca gctcactgca agctccacct cccgggttta tgccattctc ctgtctcagc 961 ctcccgagta gctgggacta caggcacccg ccaacacgcc cggctaattt ttcgtatttt1021 tagtaaagac agggtttcac cgtgttagcc aggatggtct ctatctcctg acctcgtgat1081 ctgcctgcct tggccttatt attttttttt tttaaggaca gagtctctct ctgtcaccca1141 ggctggagtg caatggcgcg atcttggctc actgtaactt ccacttgcca ggctcaagca1201 gttctcctgc ctcagcctcc tgagtagctg ggactacagg cacccgccac catgcccagc1261 taatttttgt atttttagta gagacagagt ttcaccatat tagcctggct ggtctcaaac1321 tcctggcctc aggtgatctg cccacctcgg cctcccaaag tgctgggatc aaatccactg1381 ttaatcatta ggctgaactg tctcttatag aatgaggtca aagacactcc cagttgcagg1441 gagggtagat ggccccaccc agaccgagag acacagtgat gacctcagcc tagggacacc1501 aaaaaaaaaa aaaaaaaaaa cccaaaccaa aaacgcaaac caaagcaggc aggcagacag1561 ctgctggggg aaatcctggg gtccttgaga cagaggcagg accctcgtgt tcccagctgc1621 ctcttgcctt gatagtggtg ctgtgtccct ctcagacccc ccacctgagt ctccacagag1681 ccccacgcct ggcatggcat tccacagaaa ccataaaggt tggctgagtc cIt is also expected that Coco-related proteins also exist in otherspecies, including family members in Xenopus, and Drosophila, C.elegans, zebrafish, as well as in all mammals, for example, rats, miceand non-human primates. “Coco or Coco-like proteins” also includesvariants of the naturally occurring Coco proteins, such as allelicvariants or variants induced by mutagenesis or deletions, and fragmentsof Coco proteins which variants and fragments retain myostatin bindingactivity. “Coco-like” proteins is also used to signify the family ofproteins sharing structural and/or functional similarity, includingthose proteins which are described further herein. Such proteins mayhave amino acid sequences sharing significant sequence identity (e.g.,at least about 50%, 60%, 70%, 80%, 90%, 95%, 99% or more) with the humanCoco protein, over the full-length, or at least within the myostatinbinding domain of the human Coco. Coco-like proteins also includeproteins that have amino acid sequences that are encoded by nucleic acidsequences that hybridize under stringent conditions with the codingsequences for human Coco, particularly that portion of the codingsequence for the myostatin binding domain. A Coco derivative or variantsequence may or may not lack the N-terminal BMP binding domain.

Unless specifically stated otherwise, “Cerberus (derivative)therapeutics” or its grammatical variations include the full-length orthe N-terminally truncated versions of Cerberus therapeutics.

As used herein, the term “Cerberus or Cerberus-like activity” refers toone or more of the activities which are exhibited by the mammalianCerberus-like proteins of the present disclosure. In particular,“Cerberus or Cerberus-like activity” includes the ability to induce,enhance and/or inhibit the formation, growth, proliferation,differentiation, maintenance of neurons and/or related neural cells andtissues such as brain cells, Schwann cells, glial cells and astrocytes.“Cerberus or Cerberus-like” activity also includes the ability to inducemolecular markers of neuroendocrine or ectoderm tissue, such as OTX2,N-CAM, MASH, chromagranin, and AP2, as well as the ability to induce theformation of neurons and/or related neural cells and tissues such asbrain cells, Schwann cells, glial cells and astrocytes. “Cerberus orCerberus-like activity” may also include the ability to regulate theinteraction of ligands and their protein receptors. “Cerberus orCerberus-like activity” may further include the ability to regulate theformation, differentiation, proliferation and/or maintenance of othercells and/or tissue, for example connective tissue, organs and woundhealing. In particular, “Cerberus or Cerberus-like activity” may includethe ability to enhance and/or inhibit the formation, growth,proliferation, differentiation and/or maintenance of cardiac, spleen,liver, pancreas, stomach, kidney, lung and brain cells and tissue, aswell as osteoblasts and bone, chondrocytes and cartilage, tendon,epidermis and muscle. “Cerberus and Cerberus-like activity” alsoincludes the activities of Cerberus and Cerberus-like protein in theassays described in the examples and specification herein.

Cerberus and Cerberus-like nucleotide sequences in mouse and human areas disclosed in US 2002/0164682 A1, as SEQ ID NOs. 1 and 7 (incorporatedherein by reference). Also see NCBI RefSeq ID NM_(—)005454.1 (human) andNM_(—)009887.1 (mouse).

In certain related embodiments, the mysotatin inhibitor is a polypeptidethat includes a myostatin binding domain of a Coco protein, such as thehuman Coco protein.

The terms “antibody” and “antibody agent” are used interchangeablyherein, and refer to an immunoglobulin molecule obtained by in vitro orin vivo generation of the humoral response, and includes both polyclonaland monoclonal antibodies. The term also includes genetically engineeredforms such as chimeric antibodies (e.g., humanized murine antibodies),heteroconjugate antibodies (e.g., bispecific antibodies), andrecombinant single chain Fv fragments (scFv). The term “antibody” alsoincludes antigen binding forms of antibodies (e.g., Fab′, F(ab′)₂, Fab,Fv, rIgG, and, inverted IgG).

The term “antigen binding fragment” includes any portion of an antibodythat binds to a target epitope. An antigen binding fragment may be, forexample, a polypeptide including a CDR3 region, or other fragment of animmunoglobulin molecule which retains the affinity and specificity ofthe myostatin epitope.

“Specifically binds” includes reference to the preferential associationof a ligand, in whole or part, with a particular target molecule (i.e.,“binding partner” or “binding moiety”) relative to compositions lackingthat target molecule. It is, of course, recognized that a certain degreeof non-specific interaction may occur between the subject myostatinneutralizing antibodies and a other proteins. Nevertheless, specificbinding, may be distinguished as mediated through specific recognitionof the myostatin protein. Typically specific binding results in a muchstronger association between the antibody and myostatin protein thanbetween the antibody and other proteins, e.g., GDF11. Specific bindingby an antibody to myostatin under such conditions requires an antibodythat is selected for its specificity for a particular protein. Theaffinity constant (Ka, as opposed to Kd) of the antibody binding sitefor its cognate monovalent antigen is at least 10⁷, usually at least10⁸, preferably at least 10⁹, more preferably at least 10¹⁰, and mostpreferably at least 10¹¹M. A variety of immunoassay formats areappropriate for selecting antibodies specifically reactive withmyostatin. For example, solid-phase ELISA immunoassays are routinelyused to select monoclonal antibodies specifically reactive with aprotein. See Harlow and Lane (1988) Antibodies, A Laboratory Manual,Cold Spring Harbor Publications, New York, for a description ofimmunoassay formats and conditions that can be used to determinespecific reactivity.

Immunoassays in the competitive binding format can be used to determinecross-reactivity of antibodies with myostatin, e.g., to identify whethera test antibody is a myostatin neutralizing antibody. For example, themyostatin protein, or a fragment thereof is immobilized to a solidsupport. Test antibodies are added to the assay compete with the bindingof a TGF receptor, such as ActRII or ALK7, to the immobilized antigen.The ability of the test antibodies to compete with the binding of a TGFreceptor to the immobilized myostatin antigen is compared.

Similarly, immunoassays in the competitive binding format can be used todetermine cross-reactivity determinations, e.g., to determine thespecificity of a myostatin neutralizing antibody. For example, themyostatin protein, or the myostatin epitope thereof is immobilized to asolid support. Epitopes from other proteins, such as 11, Nodal or BMP-4or other proteins having sequence homology with myostatin are added tothe assay to compete with the binding of a potential myostatinneutralizing antibody to the immobilized antigen. The ability of thetest peptides to compete with the binding of potential myostatinneutralizing antibody with the immobilized myostatin antigen iscompared. The percent cross-reactivity of the potential myostatinneutralizing antibody for the other antigens is calculated, usingstandard calculations. In certain preferred embodiments, the subjectmyostatin neutralizing antibodies have less than 10% cross-reactivitywith 11. In other preferred embodiments, the subject myostatinneutralizing antibodies have less than 1%, 5%, or 10% cross-reactivitywith BMP-4.

III. Exemplary Cerberus and Coco Derivatives

In certain embodiments, the mysotatin inhibitor is a Cerberuspolypeptide sharing at least about 50%, 60%, 70%, 80%, 90%, 95%, 99% ormore sequence identity over the full-length of the human or mouseCerberus protein.

In certain other embodiments, the mysotatin inhibitor is a polypeptidethat includes a Cerberus sequence obtained from human, mouse, or otherspecies, their variants or derivatives, including N-terminally truncatedversions of Cerberus. The full-length mouse and human Cerberus proteins,disclosed as SEQ ID NOs. 2 and 8, respectively, in US 2002/0164682 A1,are also disclosed in NCBI RefSeq format below:

Human Cerberus full length protein (SEQ ID NO: 2): 1 MHLLLFQLLVLLPLGKTTRH QDGRQNQSSL SPVLLPRNQR ELPTGNHEEA EEKPDLFVAV 61 PHLVATSPAGEGQRQREKML SRFGRFWKKP EREMHPSRDS DSEPFPPGTQ  SLIQPIDGMK 121 MEKSPLREEAKKFWHHFMFR KTPASQGVIL PIKSHEVHWE TCRTVPFSQT ITHEGCEKVV 181 VQNNLCFGKCGSVHFPGAAQ HSHTSCSHCL PAKFTTMHLP LNCTELSSVI KVVMLVEECQ 241 CKVKTEHEDG HILHAGSQDS  FIPGVSA

Residues 106-119 (from any one of which residues the subject Cerberusderivatives may begin), and residues 241-267 (to any one of whichresidues the subject Cerberus derivatives may end) are underlined.

Mouse Cerberus full length protein (SEQ ID NO: 1): 1 MHLLLVQLLVLLPLGKADLC VDGCQSQGSL SFPLLERGRR DLHVANHEEA EDKPDLFVAV 61 PHLNGTSLAGEGQRQRGKML SRLGRFWKKP ETEFYPPRDV ESDHVSSGMQ  AVTQPADGRK 121 VERSPLQEEAKRFWHRFMFR KGPAFQGVIL PIKSHEVHWE TCRTVPFNQT IAHEDCQKVV 181 VQNNLCFGKCSSIRFPGEGA DAHSFCSHCS PTKFTTVHLM LNCTSPTPVV KMVMQVEECQ 241 CMVKTERGEE RLLLAGSQGS  FIPGLPASKT  NP

Residues 106-119 (from any one of which residues the subject Cerberusderivatives may begin), and residues 241-272 (to any one of whichresidues the subject Cerberus derivatives may end) are underlined. Notethat the mouse protein is largely homologous to the human proteinthroughout the sequences, with the exception of 5 additional residues atthe C-terminus. Therefore, whenever a non-human Cerberus derivative isused, the residue numbers refers to those corresponding to the humansequences.

As described above, in certain embodiments, preferred fragments of thehuman Cerberus derivative proteins are ones which begins anywhere fromresidues 106-119 (inclusive) at the N-terminus, and ends anywhere afterresidue 241. A variety of additional Cerberus and Coco derivatives andvariants are described in the Examples.

Also included are Cerberus derived variant sequence, including mutantsor variants of the wild-type myostatin binding domains that retainmyostatin binding activity, optionally substantially loses BMP-4binding. Variant sequences without BMP binding affinity may be desirableas a way to alter selectivity of the inhibitor (e.g., relative to 11 ornodal binding, where preferential binding to one of the proteins occur.Also includes more preferential—higher affinity than wild-type—bindingto myostatin, or more discrimitory—lower affinity than wild-typetruncated version—binding to BMP-4), alter other binding characteristicswith respect to myostatin (such as K_(d), and/or K_(on) or K_(off)rates), or improve biodistribution or half life in vivo or on the shelf.

Certain other Cerberus sequences are listed below based on homologysearch in databases of identified proteins, and the subject variantCerberus polypeptides can be derived from those proteins as well. Sincethese sequences are retrieved from public databases available on theinternet, additional homologs of the proteins in other species may beobtained as these databases are being updated. Furthermore, otherspecies of Cerberus proteins, especially those of mammals, can bereadily obtained by standard molecular biology protocols, such as PCR,low stringency hybridization, Ab-mediated screening of expressionlibraries using antibodies cross-reacting with identified Cerberushomologs in target species, etc.

For example, sequence alignments using softwares such as DNAStar'sMegaAlign (supra) can identify the most conserved regions in the knownmembers of a protein family. PCR can then be carried out usingdegenerate oligoes covering such most conserved regions, and templatesDNA from the target organism. In preferred embodiments, such conservedregions include the kinase domain, and/or the ligand binding domain.

These same conserved regions may be used to generate probes forscreening nucleic acid libraries at moderate to low stringencyhybridization conditions (see definition section).

In certain embodiments, the mysotatin inhibitor is a Cerberuspolypeptide sharing at least about 50%, 60%, 70%, 80%, 90%, 95%, 99% ormore sequence identity over the full-length of the human or mouseCerberus protein.

In certain other embodiments, the mysotatin inhibitor is a polypeptidethat includes a Coco sequence obtained from human, mouse, or otherspecies, their variants or derivatives, including N-terminally truncatedversions of Coco. The full-length human Coco protein is disclosed above.

The various Cerberus and Coco polypeptides may be prepared as fusionproteins. A fusion protein may include one or more additionalpolypeptide portion that enhance one or more of in vivo stability, invivo half life, uptake/administration, tissue localization ordistribution, formation of protein complexes, and/or purification. Forexample, a fusion protein may include a portion of a constant region ofa immunoglobulin heavy chains, e.g., an immunoglobulin Fc domain, and/ora purification subsequence selected from: an epitope tag, a FLAG tag, apolyhistidine sequence, and a GST fusion. The myostatin antagonistprotein may include one or more modified amino acid residues selectedfrom: a glycosylated amino acid, a PEGylated amino acid, a farnesylatedamino acid, an acetylated amino acid, a biotinylated amino acid, anamino acid conjugated to a lipid moiety, and an amino acid conjugated toan organic derivatizing agent.

A fusion protein or coupled protein system (e.g. non-fusion covalentlinkage by crosslinking) may also include a second myostatin inhibitordomain, which is a polypeptide affinity reagent that selectively bindsto myostatin and competes with the binding of an ALK7 or ALK4 receptor.The affinity reagent may be an antibody agent. An antibody agent may be,for example, a recombinant antibody; a monoclonal antibody; a VH domain;a VL domain; an scFv; an Fab fragment; an Fab′ fragment; an F(ab′)2; anFv; or a disulfide linked Fv, a fully human antibody or a humanizedchimeric antibody, or an antigen binding fragment thereof. An affinityreagent is a peptide or scaffolded peptide that selectively binds tomyostatin and competes with the binding of an ALK7 or ALK4 receptor. Anaffinity reagent may include a myostatin binding domain of ALK7 or ALK4.For example, an extracellular domain of ALK7 or ALK4 (preferably humanALK7 or ALK4) may be used. The affinity reagent may be a small organicmolecule that selectively binds to myostatin and competes with thebinding of an ALK7 or ALK4 receptor.

An example of a human ALK7 myostatin binding domain is shown below:

(SEQ ID NO: 9) LKCVCLLCDSSNFTCQTEGACWASVMLTNGKEQVIKSCVSLPELNAQVFCHSSNNVTKTECCFTDFCNNITLHLP

An example of a human ALK4 myostatin binding domain is shown below:

(SEQ ID NO: 10) ALLCACTSCLQANYTCETDGACMVSIFNLDGMEHHVRTCIPKVELVPAGKPFYCLSSEDLRNTHCCYTDY

As shown herein, Caronte, the chicken ortholog of Cerberus does notsubstantially inhibit Activin A signaling in an A204 Reporter GeneAssay. Similarly, we have determined that human Cerberus and Coco do notinhibit Activin A. Thus, such myostatin antagonists will preferablyexhibit little or no interaction with Activin A-mediated signaling.

IV. Exemplary Therapeutic Uses

The subject Coco and Cerberus polypeptides, such as the full-length andthe N-terminally truncated Cerberus derivatives or Coco derivatives, canbe used in a number of therapeutic settings to treat a number ofdiseases resulting from or exacerbated by the presence of myostatin.Decreased myostatin expression or activity has been shown to bebeneficial for promoting muscle growth, inhibiting fat accumulation andnormalizing glucose homeostasis in the context of models of diabetes.

In certain embodiments, the subject polypeptides and derivatives thereofare used as part of a treatment for a muscular dystrophy. The term“muscular dystrophy” refers to a group of degenerative muscle diseasescharacterized by gradual weakening and deterioration of skeletal musclesand sometimes the heart and respiratory muscles. Muscular dystrophiesare genetic disorders characterized by progressive muscle wasting andweakness that begin with microscopic changes in the muscle. As musclesdegenerate over time, the person's muscle strength declines. Exemplarymuscular dystrophies that can be treated with a regimen including thesubject myostatin include: Duchenne Muscular Dystrophy (DMD), BeckerMuscular Dystrophy (BMD), Emery-Dreifuss Muscular Dystrophy (EDMD),Limb-Girdle Muscular Dystrophy (LGMD), Facioscapulohumeral MuscularDystrophy (FSH or FSHD) (Also known as Landouzy-Dejerine), MyotonicDystrophy (MMD) (Also known as Steinert's Disease), OculopharyngealMuscular Dystrophy (OPMD), Distal Muscular Dystrophy (DD), CongenitalMuscular Dystrophy (CMD).

Duchenne Muscular Dystrophy (DMD) was first described by the Frenchneurologist Guillaume Benjamin Amand Duchenne in the 1860s. BeckerMuscular Dystrophy (BMD) is named after the German doctor Peter EmilBecker, who first described this variant of DMD in the 1950s. DMD is oneof the most frequent inherited diseases in males, affecting one in 3,500boys. DMD occurs when the dystrophin gene, located on the short arm ofthe X chromosome, is broken. Since males only carry one copy of the Xchromosome, they only have one copy of the dystrophin gene. Without thedystrophin protein, muscle is easily damaged during cycles ofcontraction and relaxation. While early in the disease musclecompensates by regeneration, later on muscle progenitor cells cannotkeep up with the ongoing damage and healthy muscle is replaced bynon-functional fibro-fatty tissue.

In DMD, boys begin to show signs of muscle weakness as early as age 3.The disease gradually weakens the skeletal or voluntary muscles, thosein the arms, legs and trunk. By the early teens or even earlier, theboy's heart and respiratory muscles may also be affected. BMD is a muchmilder version of DMD. Its onset is usually in the teens or earlyadulthood, and the course is slower and far less predictable than thatof DMD. (Though DMD and BMD affect boys almost exclusively, in rarecases they can affect girls.

Until the 1980s, little was known about the cause of any kind ofmuscular dystrophy. In 1986, the dystrophin gene deficiency wasidentified as the cause of DMD. BMD results from different mutations inthe same gene. BMD patients have some dystrophin, but it's eitherinsufficient in quantity or poor in quality. Having some dystrophinprotects the muscles of those with BMD from degenerating as badly or asquickly as those of people with DMD.

Recent researches demonstrate that blocking or eliminating Myostatinfunction in vivo can effectively treat at least certain symptoms in DMDand BMD patients (Bogdanovich et al., supra; Wagner et al., supra).Thus, the subject Cerberus derivatives, especially the N-terminallytruncated versions thereof, constitute an alternative means of blockingthe function of Myostatin in vivo in DMD and BMD patients.

Similarly, the subject Coco or Cerberus derivatives, especially theN-terminally truncated versions thereof, provide an effective means toincrease muscle mass in other disease conditions that are in need ofmuscle growth. For example, Gonzalez-Cadavid et al. (supra) reportedthat that Myostatin expression correlates inversely with fat-free massin humans and that increased expression of the Myostatin gene isassociated with weight loss in men with AIDS wasting syndrome. Byinhibiting the function of Myostatin in AIDS patients, at least certainsymptoms of AIDS may be alleviated, if not completely eliminated, thussignificantly improving quality of life in AIDS patients.

Since loss of Myostatin function is also associated with fat losswithout diminution of nutrient intake (Zimmers et al., supra; McPherronand Lee, supra), the subject Coco or Cerberus derivatives, especiallythe N-terminally truncated versions thereof, may further be used as atherapeutic agent for slowing or preventing the development of obesityand type II diabetes.

The cancer anorexia-cachexia syndrome is among the most debilitating andlife-threatening aspects of cancer. Progressive weight loss in canceranorexia-cachexia syndrome is a common feature of many types of cancerand is responsible not only for a poor quality of life and poor responseto chemotherapy, but also a shorter survival time than is found inpatients with comparable tumors without weight loss. Associated withanorexia, fat and muscle tissue wasting, psychological distress, and alower quality of life, cachexia arises from a complex interactionbetween the cancer and the host. It is one of the most common causes ofdeath among cancer patients and is present in 80% at death. It is acomplex example of metabolic chaos effecting protein, carbohydrate, andfat metabolism. Tumors produce both direct and indirect abnormalities,resulting in anorexia and weight loss. Currently, there is no treatmentto control or reverse the process.

Cancer anorexia-cachexia syndrome affects cytokine production, releaseof lipid-mobilizing and proteolysis-inducing factors, and alterations inintermediary metabolism. Although anorexia is common, a decreased foodintake alone is unable to account for the changes in body compositionseen in cancer patients, and increasing nutrient intake is unable toreverse the wasting syndrome. Cachexia should be suspected in patientswith cancer if an involuntary weight loss of greater than five percentof premorbid weight occurs within a six-month period.

Since systemic overexpression of Myostatin in adult mice was found toinduce profound muscle and fat loss analogous to that seen in humancachexia syndromes (Zimmers et al., supra), the subject Coco or Cerberusderivatives, especially the N-terminally truncated versions thereof as apharmaceutical composition can be beneficially used as a Myostatinantagonist/blocker to prevent, treat, or alleviate the symptoms of thecachexia syndrome, where muscle growth is desired.

In certain embodiments, the subject variant Coco or Cerberuspolypeptides, particularly the N-terminally truncated Cerberusderivatives, can be used to form pharmaceutical compositions that can bebeneficially used to prevent, treat, or alleviate symptoms of a host ofdiseases involving neurodegeneration. While not wishing to be bound byany particular theory, the subject Cerberus derivatives may antagonizethe inhibitory feedback mechanism mediated through the wild-type ALK7receptor, thus allowing new neuronal growth and differentiation. Thesubject Cerberus derivative as a pharmaceutical composition can bebeneficially used to prevent, treat, or alleviate symptoms of diseaseswith neurodegeneration, including Alzheimer's Disease (AD), Parkinson'sDisease (PD), Amyotrophic Lateral Sclerosis (ALS), Huntington's disease,etc.

Alzheimer's disease (AD) is a chronic, incurable, and unstoppablecentral nervous system (CNS) disorder that occurs gradually, resultingin memory loss, unusual behavior, personality changes, and a decline inthinking abilities. These losses are related to the death of specifictypes of brain cells and the breakdown of connections between them.

AD has been described as childhood development in reverse. In mostpeople with AD, symptoms appear after the age 60. The earliest symptomsinclude loss of recent memory, faulty judgment, and changes inpersonality. Later in the disease, those with AD may forget how to dosimple tasks like washing their hands. Eventually people with AD loseall reasoning abilities and become dependent on other people for theireveryday care. Finally, the disease becomes so debilitating thatpatients are bedridden and typically develop coexisting illnesses. ADpatients most commonly die from pneumonia, 8 to 20 years from diseaseonset.

Parkinson's disease (PD) is a chronic, incurable, and unstoppable CNSdisorder that occurs gradually and results in uncontrolled bodymovements, rigidity, tremor, and gait difficulties. These motor systemproblems are related to the death of brain cells in an area of the brainthat produces dopamine—a chemical that helps control muscle activity.

In most people with PD, symptoms appear after age 50. The initialsymptoms of PD are a pronounced tremor affecting the extremities,notably in the hands or lips. Subsequent characteristic symptoms of PDare stiffness or slowness of movement, a shuffling walk, stoopedposture, and impaired balance. There are wide ranging secondary symptomssuch as memory loss, dementia, depression, emotional changes, swallowingdifficulties, abnormal speech, sexual dysfunction, and bladder and bowelproblems. These symptoms will begin to interfere with routineactivities, such as holding a fork or reading a newspaper. Finally,people with PD become so profoundly disabled that they are bedridden.People with PD usually die from pneumonia.

Amyotrophic lateral sclerosis (ALS; Lou Gehrig's disease; motor neurondisease) is a chronic, incurable CNS disorder that attacks the motorneurons, components of the CNS that connect the brain to the skeletalmuscles. In ALS, the motor neurons deteriorate and eventually die, andthough a person's brain normally remains fully functioning and alert,the command to move never reaches the muscles.

Most people are diagnosed with ALS between 40 and 70 years of age. Thefirst motor neurons that weaken are those leading to the arms or legs.Those with ALS may have trouble walking, they may drop things, fall,slur their speech, and laugh or cry uncontrollably. Eventually themuscles in the limbs begin to atrophy from disuse. This muscle weaknesswill become debilitating and a person will need a wheel chair or becomeunable to function out of bed. Most ALS patients die from respiratoryfailure or from complications of ventilator assistance like pneumonia,3-5 years from disease onset.

The causes of these neurological diseases has remained largely unknown.They are conventionally defined as distinct diseases, yet clearly showextraordinary similarities in basic processes and commonly demonstrateoverlapping symptoms far greater than would be expected by chance alone.Current disease definitions fail to properly deal with the issue ofoverlap and a new classification of the neurodegenerative disorders hasbeen called for.

Huntington's disease (HD) is another neurodegenerative disease resultingfrom genetically programmed degeneration of neurons in certain areas ofthe brain. This degeneration causes uncontrolled movements, loss ofintellectual faculties, and emotional disturbance. HD is a familialdisease, passed from parent to child through a dominant mutation in thewild-type gene. Some early symptoms of HD are mood swings, depression,irritability or trouble driving, learning new things, remembering afact, or making a decision. As the disease progresses, concentration onintellectual tasks becomes increasingly difficult and the patient mayhave difficulty feeding himself or herself and swallowing. The rate ofdisease progression and the age of onset vary from person to person.

Tay-Sachs disease and Sandhoff disease are glycolipid storage diseasescaused by the lack of lysosomal β-hexosaminidase (Gravel et al., in TheMetabolic Basis of Inherited Disease, eds. Scriver et al., McGraw-Hill,New York, pp. 2839-2879, 1995). In both disorders, G_(M2) gangliosideand related glycolipid substrates for β-hexosaminidase accumulate in thenervous system and trigger acute neurodegeneration. In the most severeforms, the onset of symptoms begins in early infancy. A precipitousneurodegenerative course then ensues, with affected infants exhibitingmotor dysfunction, seizure, visual loss, and deafness. Death usuallyoccurs by 2-5 years of age. Neuronal loss through an apoptotic mechanismhas been demonstrated (Huang et al., Hum. Mol. Genet. 6: 1879-1885,1997).

It is well-known that apoptosis plays a role in AIDS pathogenesis in theimmune system. However, HIV-1 also induces neurological disease. Shi etal. (J. Clin. Invest. 98: 1979-1990, 1996) examined apoptosis induced byHIV-1 infection of the central nervous system (CNS) in an in vitro modeland in brain tissue from AIDS patients, and found that HIV-1 infectionof primary brain cultures induced apoptosis in neurons and astrocytes invitro. Apoptosis of neurons and astrocytes was also detected in braintissue from 10/11 AIDS patients, including 5/5 patients with HIV-1dementia and 4/5 nondemented patients.

Neuronal loss is a also a salient feature of prion diseases, such asCreutzfeldt-Jakob disease in human, BSE in cattle (mad cow disease),Scrapie Disease in sheep and goats, and feline spongiform encephalopathy(FSE) in cats.

The subject Cerberus and Coco polypeptides, including the N-terminallytruncated Cerberus derivatives are also useful to prevent, treat, andalleviate symptoms of various PNS disorders, such as the ones describedbelow. The PNS is composed of the nerves that lead to or branch off fromthe CNS. The peripheral nerves handle a diverse array of functions inthe body, including sensory, motor, and autonomic functions. When anindividual has a peripheral neuropathy, nerves of the PNS have beendamaged. Nerve damage can arise from a number of causes, such asdisease, physical injury, poisoning, or malnutrition. These agents mayaffect either afferent or efferent nerves. Depending on the cause ofdamage, the nerve cell axon, its protective myelin sheath, or both maybe injured or destroyed.

The term peripheral neuropathy encompasses a wide range of disorders inwhich the nerves outside of the brain and spinal cord—peripheralnerves—have been damaged. Peripheral neuropathy may also be referred toas peripheral neuritis, or if many nerves are involved, the termspolyneuropathy or polyneuritis may be used.

Peripheral neuropathy is a widespread disorder, and there are manyunderlying causes. Some of these causes are common, such as diabetes,and others are extremely rare, such as acrylamide poisoning and certaininherited disorders. The most common worldwide cause of peripheralneuropathy is leprosy. Leprosy is caused by the bacterium Mycobacteriumleprae, which attacks the peripheral nerves of affected people.According to statistics gathered by the World Health Organization, anestimated 1.15 million people have leprosy worldwide.

Leprosy is extremely rare in the United States, where diabetes is themost commonly known cause of peripheral neuropathy. It has beenestimated that more than 17 million people in the United States andEurope have diabetes-related polyneuropathy. Many neuropathies areidiopathic—no known cause can be found. The most common of the inheritedperipheral neuropathies in the United States is Charcot-Marie-Toothdisease, which affects approximately 125,000 persons.

Another of the better known peripheral neuropathies is Guillain-Barrésyndrome, which arises from complications associated with viralillnesses, such as cytomegalovirus, Epstein-Barr virus, and humanimmunodeficiency virus (HIV), or bacterial infection, includingCampylobacter jejuni and Lyme disease. The worldwide incidence rate isapproximately 1.7 cases per 100,000 people annually. Other well-knowncauses of peripheral neuropathies include chronic alcoholism, infectionof the varicella-zoster virus, botulism, and poliomyelitis. Peripheralneuropathy may develop as a primary symptom, or it may be due to anotherdisease. For example, peripheral neuropathy is only one symptom ofdiseases such as amyloid neuropathy, certain cancers, or inheritedneurologic disorders. Such diseases may affect the peripheral nervoussystem (PNS) and the central nervous system (CNS), as well as other bodytissues.

Other PNS diseases treatable with the subject Cerberus and Cocopolypeptides include: Brachial Plexus Neuropathies (Diseases of thecervical and first thoracic roots, nerve trunks, cords, and peripheralnerve components of the brachial plexus. Clinical manifestations includeregional pain, paresthesia; muscle weakness, and decreased sensation inthe upper extremity. These disorders may be associated with trauma,including birth injuries; thoracic outlet syndrome; neoplasms, neuritis,radiotherapy; and other conditions. See Adams et al. Principles ofNeurology, 6^(th) ed, pp 1351-2); Diabetic Neuropathies (Peripheral,autonomic, and cranial nerve disorders that are associated with diabetesmellitus. These conditions usually result from diabetic microvascularinjury involving small blood vessels that supply nerves (vasa nervorum).Relatively common conditions which may be associated with diabeticneuropathy include third nerve palsy; mononeuropathy; mononeuropathymultiplex; diabetic amyotrophy; a painful polyneuropathy; autonomicneuropathy; and thoracoabdominal neuropathy. See Adams et al.,Principles of Neurology, 6^(th) ed, p 1325); Mononeuropathies (Diseaseor trauma involving a single peripheral nerve in isolation, or out ofproportion to evidence of diffuse peripheral nerve dysfunction.Mononeuropathy multiplex refers to a condition characterized by multipleisolated nerve injuries. Mononeuropathies may result from a wide varietyof causes, including ischemia; traumatic injury; compression; connectivetissue diseases; cumulative trauma disorders; and other conditions);Neuralgia (Intense or aching pain that occurs along the course ordistribution of a peripheral or cranial nerve); Peripheral NervousSystem Neoplasms (Neoplasms which arise from peripheral nerve tissue.This includes neurofibromas; Schwannomas; granular cell tumors; andmalignant peripheral nerve sheath tumors. See DeVita Jr et al., Cancer:Principles and Practice of Oncology, 5^(th) ed, pp 1750-1); NerveCompression Syndromes (Mechanical compression of nerves or nerve rootsfrom internal or external causes. These may result in a conduction blockto nerve impulses, due to, for example, myelin sheath dysfunction, oraxonal loss. The nerve and nerve sheath injuries may be caused byischemia; inflammation; or a direct mechanical effect); Neuritis (Ageneral term indicating inflammation of a peripheral or cranial nerve.Clinical manifestation may include pain; paresthesias; paresis; orhyperthesia); Polyneuropathies (Diseases of multiple peripheral nerves.The various forms are categorized by the type of nerve affected (e.g.,sensory, motor, or autonomic), by the distribution of nerve injury(e.g., distal vs. proximal), by nerve component primarily affected(e.g., demyelinating vs. axonal), by etiology, or by pattern ofinheritance).

In certain embodiments, the subject full-length Coco or Cerberuspolypeptides or variants thereof are used as part of a treatment fordiseases or conditions characterized by excessive or undesirable levelsof BMP, such as the ones described below.

The heterotopic ossification of muscles, tendons, and ligaments is acommon problem faced by orthopaedic surgeons. Hannallah et al. (J BoneJoint Surg Am. 2004 January; 86-A(1):80-91) investigated the ability ofNoggin (a BMP [bone morphogenetic protein] antagonist) to inhibitheterotopic ossification. Three varying doses of Noggin-expressingmuscle-derived stem cells inhibited the heterotopic ossificationelicited by BMP-4-expressing muscle-derived stem cells. Each of threevarying doses of Noggin-expressing muscle-derived stem cells alsosignificantly inhibited the heterotopic ossification elicited bydemineralized bone matrix. All eleven animals that underwent Achillestenotomy developed heterotopic ossification at the site of the injury inthe control limbs. In contrast, the limbs treated with theNoggin-expressing muscle-derived stem cells had a reduction in theformation of heterotopic ossification of 83% and eight of the elevenanimals had no radiographic evidence of heterotopic ossification(p<0.05). Thus, delivery of Noggin mediated by muscle-derived stem cellscan inhibit heterotopic ossification caused by BMP4, demineralized bonematrix, and trauma in an animal model, indicating that gene therapy todeliver BMP inhibitors (Noggin or Cerberus) may become a powerful methodto inhibit heterotopic ossification in targeted areas of the body. Seealso Glaser et al. (J Bone Joint Surg Am. 2003 December;85-A(12):2332-42).

Osteoarthritis (OA) is a joint disease characterized by osteophytedevelopment, fibrosis, and articular cartilage damage. Effects ofexogenous transforming growth factor beta (TGFbeta) isoforms and bonemorphogenetic proteins (BMPs) suggest a role for these growth factors inthe pathogenesis of OA. Scharstuhl et al. (Arthritis Rheum. 2003December; 48(12):3442-51) used adenoviral overexpression of TGF-beta andBMP antagonists to block the signaling of TGF-beta and BMP. Theinhibitors studied include a secreted, pan-specific TGF-beta antagonistcalled murine latency-associated peptide 1 (mLAP-1), intracellularinhibitory Smad6 (a BMP antagonist), and Smad7 (a TGF-beta/BMPinhibitor). Intraarticular injection of papain caused increased proteinexpression of several TGF-beta and BMP isoforms in synovium andcartilage. Adenovirus transfection into the joint resulted in a strongexpression of the transgenes in the synovial lining. Overexpression ofmLAP-1, Smad6, and Smad7 led to a significant reduction in osteophyteformation compared with that in controls. Smad6 and Smad7 overexpressionalso significantly decreased synovial thickening. Furthermore, thesecreted TGF-beta inhibitor mLAP-1 increased articular cartilage PGloss. These results indicate a pivotal role of excessive endogenousTGF-beta and BMP in the development of osteophytes and synovialthickening, implicating excessive endogenous TGFbeta and BMP in thepathogenesis of OA. In contrast, the prevention of cartilage damage byendogenous TGF-beta signifies the protective role of TGF-beta inarticular cartilage. Thus the subject Coco or Cerberus pharmaceuticalcompositions can be used as BMP antagonists to treat OA, including thedevelopment of osteophytes and synovial thickening.

In an analysis of normal ovarian surface epithelium (OSE) and ovariancancer (OC) cells, Shepherd and Nachtigal (Endocrinology. 2003 August;144(8):3306-14) observed BMP4 mRNA expression and found that primary OCcells produce mature BMP4. In addition, each member of the downstreamsignaling pathway was expressed in primary OSE and OC cells. Smad1 wasphosphorylated and underwent nuclear translocation in normal OSE and OCcells upon treatment with BMP4. Interestingly, the BMP target genes ID1and ID3 were up-regulated 10- to 15-fold in primary OC cells, comparedwith a 2- to 3-fold increase in normal OSE. The growth of severalprimary OC cells was relatively unaltered by BMP4 treatment; however,long-term BMP4 treatment of primary OC cells resulted in decreased celldensity as well as increased cell spreading and adherence. These datademonstrate the existence and putative function of BMP signaling innormal OSE and OC cells, and thus the subject Cerberus pharmaceuticalpreparations can be used to regulate BMP4 signaling in OC pathogenesis.

Fibrodysplasia ossificans progressiva (FOP), a rare genetic disablingdisease characterized by heterotopic bone formation, is of specialinterest for general medicine since the bone morphogenetic proteins(especially BMP4) involved in its pathogenesis are known to play a rolein skeletal morphogenesis, and the gene antagonist to BMP-4 (such asnoggin) might be useful in preventing lamellar bone formation. SeeBlaszczyk et al. (Eur J. Dermatol. 2003 May-June; 13(3):234-7). Thus thesubject Cerberus therapeutics may also be used to treat FOP.

Atherosclerosis is now viewed as an inflammatory disease occurringpreferentially in arterial regions exposed to disturbed flow conditions,including oscillatory shear stress (OS), in branched arteries. Sorescuet al. (J Biol Chem. 278(33):31128-35, 2003) suggest that BMP4 is amechanosensitive, inflammatory factor playing a critical role in earlysteps of atherogenesis in the lesion-prone areas. Thus the subjectCerberus therapeutics may be used to control BMP-4 induced inflammatoryresponse in early steps of atherogenesis in those areas.

During skull development, the cranial connective tissue frameworkundergoes intramembranous ossification to form skull bones (calvaria).As the calvarial bones advance to envelop the brain, fibrous suturesform between the calvarial plates. Expansion of the brain is coupledwith calvarial growth through a series of tissue interactions within thecranial suture complex. Craniosynostosis, or premature cranial suturefusion, results in an abnormal skull shape, blindness and mentalretardation. Recent studies have demonstrated that gain-of-functionmutations in fibroblast growth factor receptors (fgfr) are associatedwith syndromic forms of craniosynostosis. Noggin, an antagonist of bonemorphogenetic proteins (BMPs), is required for embryonic neural tube,somites and skeleton patterning. Warren et al. (Nature. 2003 Apr. 10;422(6932):625-9) show that noggin is expressed postnatally in the suturemesenchyme of patent, but not fusing, cranial sutures, and that nogginexpression is suppressed by FGF2 and syndromic fgfr signalling. Sincenoggin misexpression prevents cranial suture fusion in vitro and invivo, it is suggested that syndromic fgfr-mediated craniosynostoses maybe the result of inappropriate downregulation of noggin expression,leading to abnormally high BMP activity. Thus the subject Cerberus andCoco therapeutics may be used to down-regulate BMP activity to preventor treat such conditions.

V. Exemplary Formulations

The subject compositions may be used alone, or as part of a conjointtherapy with other compounds/pharmaceutical compositions.

The soluble Coco or Cerberus polypeptides, including the N-terminallytruncated Cerberus derivative therapeutics for use in the subjectmethods may be conveniently formulated for administration with abiologically acceptable medium, such as water, buffered saline, polyol(for example, glycerol, propylene glycol, liquid polyethylene glycol andthe like) or suitable mixtures thereof. The optimum concentration of theactive ingredient(s) in the chosen medium can be determined empirically,according to procedures well known to medicinal chemists. As usedherein, “biologically acceptable medium” includes any and all solvents,dispersion media, and the like which may be appropriate for the desiredroute of administration of the pharmaceutical preparation. The use ofsuch media for pharmaceutically active substances is known in the art.Except insofar as any conventional media or agent is incompatible withthe activity of the therapeutics, its use in the pharmaceuticalpreparation of the disclosure is contemplated. Suitable vehicles andtheir formulation inclusive of other proteins are described, forexample, in the book Remington's Pharmaceutical Sciences (Remington'sPharmaceutical Sciences. Mack Publishing Co., Easton, Pa., USA 1985).These vehicles include injectable “deposit formulations.”

Pharmaceutical formulations of the present disclosure can also includeveterinary compositions, e.g., pharmaceutical preparations of the Cocoor Cerberus derivative therapeutics suitable for veterinary uses, e.g.,for the treatment of live stock (cow, sheep, goat, pig, and horse, etc.)or domestic animals, e.g., cats and dogs.

Methods of disclosure may also be provided by rechargeable orbiodegradable devices. Various slow release polymeric devices have beendeveloped and tested in vivo in recent years for the controlled deliveryof drugs, including proteinacious biopharmaceuticals. A variety ofbiocompatible polymers (including hydrogels), including bothbiodegradable and non-degradable polymers, can be used to form animplant for the sustained release of a therapeutic at a particulartarget site.

The pharmaceutical compositions according to the present disclosure maybe administered as either a single dose or in multiple doses. Thepharmaceutical compositions of the present disclosure may beadministered either as individual therapeutic agents or in combinationwith other therapeutic agents. The treatments of the present disclosuremay be combined with conventional therapies, which may be administeredsequentially or simultaneously. The pharmaceutical compositions of thepresent disclosure may be administered by any means that enables theCoco or Cerberus derivatives to reach the targeted cells/tissues/organs.In some embodiments, routes of administration include those selectedfrom the group consisting of oral, intravesically, intravenous,intraarterial, intraperitoneal, local administration into the bloodsupply of the organ in which the targeted cells reside or directly intothe cells. Intravenous administration is the preferred mode ofadministration. It may be accomplished with the aid of an infusion pump.

The phrases “parenteral administration” and “administered parenterally”as used herein means modes of administration other than enteral andtopical administration, usually by injection, and includes, withoutlimitation, intravenous, intramuscular, intraarterial, intrathecal,intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,transtracheal, subcutaneous, subcuticular, intraarticulare, subcapsular,subarachnoid, intraspinal and intrastermal injection and infusion.

The phrases “systemic administration,” “administered systemically,”“peripheral administration” and “administered peripherally” as usedherein mean the administration of a compound, drug or other materialother than directly into the central nervous system, such that it entersthe patient's system and, thus, is subject to metabolism and other likeprocesses, for example, subcutaneous administration.

These compounds may be administered to humans and other animals fortherapy by any suitable route of administration, including orally,intravesically, nasally, as by, for example, a spray, rectally,intravaginally, parenterally, intracistemally and topically, as bypowders, ointments or drops, including buccally and sublingually.

Regardless of the route of administration selected, the compounds of thepresent disclosure, which may be used in a suitable hydrated form,and/or the pharmaceutical compositions of the present disclosure, areformulated into pharmaceutically acceptable dosage forms such asdescribed below or by other conventional methods known to those of skillin the art.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions of this disclosure may be varied so as to obtain an amountof the active ingredient which is effective to achieve the desiredtherapeutic response for a particular patient, composition, and mode ofadministration, without being toxic to the patient.

The selected dosage level will depend upon a variety of factorsincluding the activity of the particular compound of the presentdisclosure employed, or the ester, salt or amide thereof, the route ofadministration, the time of administration, the rate of excretion of theparticular compound being employed, the duration of the treatment, otherdrugs, compounds and/or materials used in combination with theparticular therapeutic employed, the age, sex, weight, condition,general health and prior medical history of the patient being treated,and like factors well known in the medical arts.

A physician or veterinarian having ordinary skill in the art can readilydetermine and prescribe the effective amount of the pharmaceuticalcomposition required. For example, the physician or veterinarian couldstart doses of the compounds of the disclosure employed in thepharmaceutical composition at levels lower than that required in orderto achieve the desired therapeutic effect and gradually increase thedosage until the desired effect is achieved.

In general, a suitable daily dose of a compound of the disclosure willbe that amount of the compound which is the lowest dose effective toproduce a therapeutic effect. Such an effective dose will generallydepend upon the factors described above. Generally, intravenous,intracerebroventricular and subcutaneous doses of the compounds of thisdisclosure for a patient will range from about 0.0001 to about 100 mgper kilogram of body weight per day.

If desired, the effective daily dose of the active compound may beadministered as two, three, four, five, six or more sub-dosesadministered separately at appropriate intervals throughout the day,optionally, in unit dosage forms.

The term “treatment” is intended to encompass also prophylaxis, therapyand cure.

The patient receiving this treatment is any animal in need, includingprimates, in particular humans, and other non-human mammals such asequines, cattle, swine and sheep; and poultry and pets in general.

The compound of the disclosure can be administered as such or inadmixtures with pharmaceutically acceptable carriers and can also beadministered in conjunction with other antimicrobial agents such aspenicillins, cephalosporins, aminoglycosides and glycopeptides.Conjunctive therapy, thus includes sequential, simultaneous and separateadministration of the active compound in a way that the therapeuticaleffects of the first administered one is not entirely disappeared whenthe subsequent is administered.

Combined with certain formulations, the subject Coco or Cerberusderivatives can be effective soluble agents. The therapeutic polypeptidecan be provided a fusion peptide along with a second peptide whichpromotes solubility. To illustrate, the Cerberus derivatives of thepresent disclosure can be provided as part of a fusion polypeptide withall or a fragment of the hinge or Fc portion of the immunoglobulin,which can promote solubility and/or serum stability.

The present disclosure also contemplates a peptidomimetic sequence ofthe subject polypeptide derivatives as described herein.

Generally, the nomenclature used herein and the laboratory proceduresutilized in the present disclosure include molecular, biochemical,microbiological and recombinant DNA techniques. Such techniques arethoroughly explained in the literature. See, for example, “MolecularCloning: A laboratory Manual” Sambrook et al., (1989); “CurrentProtocols in Molecular Biology” Volumes I-III Ausubel, R. M., ed.(1994); Ausubel et al., “Current Protocols in Molecular Biology”, JohnWiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guide toMolecular Cloning”, John Wiley & Sons, New York (1988); Watson et al.,“Recombinant DNA”, Scientific American Books, New York; Birren et al.(eds) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, ColdSpring Harbor Laboratory Press, New York (1998); methodologies as setforth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis,J. E., ed. (1994); “Current Protocols in Immunology” Volumes I-IIIColigan J. E., ed. (1994); Stites et al. (eds), “Basic and ClinicalImmunology” (8^(th) Edition), Appleton & Lange, Norwalk, Conn. (1994);Mishell and Shiigi (eds), “Selected Methods in Cellular Immunology”,W.H. Freeman and Co., New York (1980); available immunoassays areextensively described in the patent and scientific literature, see, forexample, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578;3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533;3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521;“Oligonucleotide Synthesis” Gait, M. J., ed. (1984); “Nucleic AcidHybridization” Hames, B. D., and Higgins S. J., eds. (1985);“Transcription and Translation” Hames, B. D., and Higgins S. J., eds.(1984); “Animal Cell Culture” Freshney, R. I., ed. (1986); “ImmobilizedCells and Enzymes” IRL Press, (1986); “A Practical Guide to MolecularCloning” Perbal, B., (1984) and “Methods in Enzymology” Vol. 1-317,Academic Press; “PCR Protocols: A Guide To Methods And Applications”,Academic Press, San Diego, Calif. (1990); Marshak et al., “Strategiesfor Protein Purification and Characterization—A Laboratory CourseManual” CSHL Press (1996); all of which are incorporated by reference asif fully set forth herein. Other general references are providedthroughout this document. The procedures therein are believed to be wellknown in the art and are provided for the convenience of the reader. Allthe information contained therein is incorporated herein by reference.

EXEMPLIFICATION

The disclosure now being generally described, it will be more readilyunderstood by reference to the following examples, which are includedmerely for purposes of illustration of certain embodiments andembodiments of the present disclosure, and are not intended to limit thedisclosure.

Example 1 Sources of Caronte and Human Cerberus Protein

Caronte-Fc (Cerberus homolog from Gallus gallus) was ordered from R&DSystems (Minneapolis, Minn.).

Full-length and N-terminally truncated forms of human Cerberus sequencewere cloned into a human CMV derived expression vector, either with orwithout a C-terminal fusion to an Fc portion of IgG1 (both human andmurine IgG1 Fc fusions were produced). These constructs were transientlytransfected in HEK293 cells using polyethylenimine (PEI). Afterculturing, cells were harvested and conditioned media was collected forpurification.

The following constructs were tested:

Human Cerberus, full length, no Fc (SEQ ID NO: 11) MHLLLFQLLV LLPLGKTTRHQDGRQNQSSL SPVLLPRNQR ELPTGNHEEA EEKPDLFVAV PHLVATSPAG EGQRQREKMLSRFGRFWKKP EREMHPSRDS DSEPFPPGTQ SLIQPIDGMK MEKSPLREEA KKFWHHFMFRKTPASQGVIL PIKSHEVHWE TCRTVPFSQT ITHEGCEKVV VQNNLCFGKC GSVHFPGAAQHSHTSCSHCL PAKFTTMHLP LNCTELSSVI KVVMLVEECQ CKVKTEHEDG HILHAGSQDSFIPGVSA

Human Cerberus, full length, Fc (TGGG linker (SEQ ID NO: 27) and Fc,underlined; native signal sequence underlined with dotted line) (SEQ IDNO:12)

Human Cerberus, short form, Fc (TGGG linker (SEQ ID NO: 27) and Fc,underlined) (SEQ ID NO:13)

EVHWETCRTV PFSQTITHEG CEKVVVQNNL CFGKCGSVHF PGAAQHSHTS CSHCLPAKFTTMHLPLNCTE LSSVIKVVML VEECQCKVKT EHEDGHILHA GSQDSFIPGV SA TGGGTHTCPPCPAPELLGGP  SVFLFPPKPK  DTLMISRTPE  VTCVVVDVSH  EDPEVKFNWY  VDGVEVHNAKTKPREEQYNS  TYRVVSVLTV  LHQDWLNGKE  YKCKVSNKAL  PVPIEKTISK  AKGOPREPQVYTLPPSREEM  TKNQVSLTCL  VKGFYPSDIA  VEWESNGQPE  NNYKTTPPVL  DSDGSFFLYSKLTVDKSRWQ  QGNVFSCSVM  HEALHNHYTQ  KSLSLSPGK

Three different leader sequences were considered:

  (i) Honey bee mellitin (HBML): MKFLVNVALVFMVVYISYIYA (SEQ ID NO: 14) (ii) Tissue Plasminogen Activator (TPA): MDAMKRGLCCVLLLCGAVFVSP (SEQ IDNO: 15) (iii) Native: MHLLLFQLLV LLPLGKT. (SEQ ID NO: 16)

A heterologous or native leader sequence may be fused to the proteinsequence at any position within the first 30 amino acids. Modelingsuggests that the native leader would yield a product beginning with“TRH . . . ” at position 18. Analysis of products expressed hereinindicates that the native leader more typically yields a productbeginning “KTT . . . ” at position 16. Therefore, heterologous leadersequences may be fused N-terminal to position 16 or position 18.

Example 2 Caronte Binds 11

11 is a close homolog of myostatin that regulates neurologicalprocesses. 11 was immobilized on a BiaCore CM5 chip using standard aminecoupling procedure. Trace: Caronte (200 μg/ml; R&D Systems) was injectedon the 11 coupled chip. The tracing in FIG. 2 shows binding of Caronteto 11.

Example 3 Caronte and Human Cerberus Inhibit 11 and Myostatin-MediatedSignaling

An A-204 Reporter Gene Assay was used to evaluate the effects of Caronteand Cerberus on signaling by 11, myostatin and Activin A. Cell line:Human Rhabdomyosarcoma (derived from muscle). Reporter vector:pGL3(CAGA)12 (Described in Dennler et al, 1998, EMBO 17: 3091-3100.) SeeFIG. 3. The CAGA12 motif is present in TGF-Beta responsive genes (PAI-1gene), so this vector is of general use for factors signaling throughSmad2 and 3.

Day 1: Split A-204 cells into 48-well plate.

Day 2: A-204 cells transfected with 10 ug pGL3(CAGA)12 orpGL3(CAGA)12(10 ug)+pRLCMV (1 ug) and Fugene.

Day 3: Add factors (diluted into medium+0.1% BSA). Inhibitors need to bepreincubated with Factors for 1 hr before adding to cells. 6 hrs later,cells rinsed with PBS, and lyse cells.

This is followed by a Luciferase assay. In the absence of anyinhibitors, Activin A showed 10 fold stimulation of reporter geneexpression and an ED50˜2 ng/ml. GDF-8: ED50: ˜5 ng/ml, 15 foldstimulation. 11: 16 fold stimulation, ED50: ˜1.5 ng/ml.

As shown in FIG. 4, Caronte inhibits 11 signaling in the A-204 ReporterGene Assay. An ActRIIA-Fc (“IIA muG2a”) fusion also inhibits 11signaling. As shown in FIG. 5, Caronte does not inhibit Activin A in theA-204 Reporter Gene Assay. An ActRIIA-Fc fusion (“IIA muG2a”), asexpected, does inhibit Activin A signaling. Thus, Caronte is a selectiveinhibitor of 11/myostatin while not affecting Activin A signaling. Thistype of selectivity suggests that Caronte, Cerberus and Coco will haverelatively few side effects when used as a therapeutic. As expected,Cerberus behaved much like Caronte, and inhibited myostatin signaling.See FIG. 6. Similar experiments were conducted to test the binding ofhuman Cerberus and Coco to Activin A, and these experiments confirm thatthese molecules do not bind to Activin A.

Example 4 Sources of Human Coco Protein

Full-length human Coco was cloned into a human CMV derived expressionvector, either with or without a C-terminal fusion to an Fc portion ofIgG1 (both human and murine IgG1 Fc fusions were produced). Theseconstructs were transiently transfected in HEK293 cells usingpolyethylenimine (PEI). After culturing, cells were harvested andconditioned media was collected for purification.

The following construct was made, and a murine Fc fusion was made also,and used in the assays presented herein:

Human Coco, full length, Fc (TGGG linker and mFc) (SEQ ID NO: 17)MLLGQLSTLL CLLSGALPTG SGRPEPQSPR PQSWAAANQT WALGPGALPP LVPASALGSWKAFLGLQKAR QLGMGRLQRG QDEVAAVTLP LNPQEVIQGM CKAVPFVQVF SRPGCSAIRLRNHLCFGNCS SLYIPGSDPT PLVLCNSCMP ARKRWAPVVL WCLTGSSASR RRVKISTMLIEGCHCSPKA TGGGTHTCPP  CPAPELLGGP  SVFLFPPKPK  DTLMISRTPE  VTCVVVDVSHEDPEVKFNWY  VDGVEVHNAK  TKPREEQYNS  TYRVVSVLTV  LHQDWLNGKE  YKCKVSNKALPVPIEKTISK  AKGQPREPQV  YTLPPSREEM  TKWQVSLTCL  VKGFYPSDIA  VEWESNGQPENNYKTTPPVL  DSDGSFFLYS  KLTVDKSRWQ  QGNVFSCSVM  HEALHNHYTQ  KSLSLSPGK

The following construct is analogous to the short form Cerberus-Fcfusion protein:

Human Coco, short form, Fc (TGGG linker and mFc) (SEQ ID NO: 18)LNPQEVIQGM CKAVPFVQVF SRPGCSAIRL RNHLCFGHCS SLYIPGSDPT PLVLCNSCMPARKRWAPVVL WCLTGSSASR RRVKISTMLI EGCNCSPKA TGGGTHTCPP  CPAPELLGGP SVFLFPPKPK  DTLMISRTPE  VTCVVVDVSH EDPEVKFNWY  VDGVEVHNAK  TKPREEQYNS TYRVVSVLTV  LHQDWLNGKE  YKCKVSNKAL PVPIEKTISK  AKGQPREPQV  YTLPPSREEM TKNQVSLTCL  VKGFYPSDIA  VEWESNGQPE NNYKTTPPVL  DSDGSFFLYS  KLTVDKSRWQ QGNVFSCSVM  HEALHNHYTQ  KSLSLSPGK

Three different leader sequences were considered:

  (i) Honey bee mellitin (HBML): MKFLVNVALVFMVVYISYIYA (SEQ ID NO: 14) (ii) Tissue Plasminogen Activator (TPA): MDAMKRGLCCVLLLCGAVFVSP (SEQ IDNO: 15) (iii) Native: MLLGQLSTLL CLLSGALPTG S. (SEQ ID NO: 19)

Heterologous or native leader sequences may be fused anywhere in thefirst 30 amino acids, and particularly N-terminal to any of amino acids16-23.

Example 5 Human Coco Inhibits 11 Signaling

Conditioned medium from cells expressing human Coco-mFc was tested foreffects on A-204 reporter gene expression in the presence of 11. Asshown in FIG. 8, conditioned medium containing Coco-mFc inhibits 11signaling in the A-204 Reporter Gene Assay, much like Cerberus. Similarexperiments showed that Coco-mFc inhibits Nodal signaling.

Example 6 Human Cerberus-Fc is Degraded in Human Serum

The stability of Cerberus polypeptides in the presence of serum wasevaluated. Conditioned medium from cells expressing human full-lengthCerberus-Fc was incubated overnight at 37 deg. C. with varying amountsof human serum (percentages of serum added are shown at top), andresolved by SDS-PAGE. Western blot (FIG. 7) showed that Cerberus wascompletely degraded when incubated with 5% human serum.

N-terminal sequencing of cleavage fragments revealed that proteolysisoccurred at the following sites (cleavage shown by ^):

38 NQR{circumflex over ( )}ELP 43 (SEQ ID NO: 24) 138 MFR{circumflexover ( )}KTP 143 (SEQ ID NO: 23) 207 SHC{circumflex over ( )}LPA 212(SEQ ID NO: 28)

Example 7 Serum Stable Human Cerberus and Coco Polypeptides

To produce serum-stable Cerberus polypeptides, a variety of mutationsmay be introduced at cleavage sites and the surrounding sequences. Inshort forms of Cerberus, only the L212 cleavage site remains (amino acidnumbering is with reference to the full length, native Cerberussequence, SEQ ID NO: 2), and so a mutation of any, some or all of theamino acids in the sequence SHCLPA (SEQ ID NO: 28) may be altered toeliminate this cleavage site. Generally, mutations will be to small,uncharged groups, such as alanine or serine. Mutations of C211 and/orL212 to serine or alanine are particularly desirable. In addition, or inthe alternative, an N-linked glycosylation site (NXT/S) may beintroduced at a position within the range of amino acids 202-222. AnN-linked glycosylation site may also be introduced at a position that isexpected to be proximal to the 212 position in the three-dimensionalstructure of the protein. Similar mutations may be made at each of theother sites 38 NQR^ELP 43 (SEQ ID NO: 24) and 138 MFR^KTP 143 (SEQ IDNO: 23), depending on the length of the Cerberus molecule to beemployed. A particularly desirable mutation with respect to the 38NQR^ELP 43 (SEQ ID NO: 24) cleavage site is an R to S/T mutation to makethe sequence 38 NQ(S/T)ELP 43 (SEQ ID NO: 29), simultaneouslyeliminating the cleavage site and introducing an N-linked glycosylationsite. Additionally, experiments have shown that products cleaved at E41and K141 retain myostatin binding activity. Accordingly, N-terminallytruncated forms of Cerberus, beginning at E41 or K141 will be resistantto cleavage at these sites and retain activity. The activity of theshort form suggests that a minimal myostatin binding domain is thecysteine knot, located at amino acids 162-241 of SEQ ID NO:2.

Cerberus constructs with one or more of the alterations (shown inbrackets below; e.g., “[R(T)]” means that an arginine normally at theposition may be replaced with a threonine) will have N-linkedglycosylation sites that will block cleavage and are expected to conferimproved pharmacokinetic properties. The constructs below may beexpressed, for example, with a tPA leader sequence and an Fc sequence.

(SEQ ID NO: 20) TRHQDGRQNQSSLSPVLLPRNQ[R(T)]ELPTGNHEEAEEKYDLFVAVPHLVATSPAGEGQRQREKMLSRFGRFWKKPEREMHPSRDSDSEPFPPGTQSLIQPIDGMKMEKSPLREEAKKFWHHFMF[R(N)]KTPASQGVILPIKSHEVHWETCRTVPFSQTITHEGCEKVVVQNNLCFGKCGSVHFPGAAQHSHTSCSHCLPAKFTTMHLPLNCTELSSVIKVVMLVEECQCKVKTEHEDGHILH[A(N)]GSQDSFIP[G(N)]VSATG

It is expected that Coco will behave in a manner similar to Cerberus,however, the two likely cleavage sites in Coco occur within the cysteineknot domain at the sequences: 150 PAR^KRW 155 (SEQ ID NO: 25) and 168SRR^RVK 173 (SEQ ID NO: 26). Amino acids in these positions may bealtered to eliminate the cleavage, with alanine and serine beingpreferred amino acids. In addition, or in the alternative, an N-linkedglycosylation site (NXT/S) may be introduced at or near either of thesepositions. The activity of the short form of Cerberus suggests that aminimal myostatin binding domain of Coco is the cysteine knot, locatedat amino acids 101-185 of SEQ ID NO:5.

Coco constructs with one or more of the alterations (shown in bracketsbelow) will have N-linked glycosylation sites that will block cleavageand are expected to confer improved pharmacokinetic properties. Theconstructs below may be expressed, for example, with a tPA leadersequence and an Fc sequence.

(SEQ ID NO: 21) GRPEPQSPRPQSWAAANQTWALGPGAIPPLVPASALGSWKAFLGLQKARQLGMG[R(N)]L[Q(T)]RGQDEVAAVTLPLNPQEVIQGMCKAVPFVQVFSRPGCSAIRLRNHLCFGHCSSLYIPGSDPTPLVLCNSCMPA[R(N)]K[R(T)]WAPVVLWCLTGSSASR[R(N)][R(A)][V(S)]KISTMLIEGCHC SPKA

Example 8 Cysteine Variants of Cerberus and Coco

In some proteins, odd numbers of cysteine residues result in a freesulfhydryl group that may cause protein aggregation or otherwiseinterfere with protein production. Both native Coco and native Cerberushave odd numbers of cystein residues. In order to improve the expressionof Cerberus, variants with fewer cysteine residues were generated, aswell as variants with changes in proximity to one or more of thecysteines. Relative to SEQ ID NO:2, the following variants weregenerated:

C176G;

C206G;

C223G;

N222D.

Each of these proteins were expressible and retained binding to GDF11,indicating that the biochemical activity of these proteins remainedintact. Similar specific variants may be made with respect to Coco(relative to SEQ ID NO:5):

C115G;

C145G;

C162G.

Therefore, the disclosure provides Cerberus and Coco variants in whichone or more cysteine residues are deleted or replaced. If replaced, thereplacement amino acid may be any of the other 19 canonical amino acids,although G, A, S and T are preferred.

EQUIVALENTS

A skilled artisan will recognize, or be able to ascertain using no morethan routine experimentation, many equivalents to the specificembodiments of the inventions described herein. Such equivalents areintended to be encompassed by the following claims.

1. An isolated myostatin antagonist protein, the protein comprising anamino acid sequence that has at least 90% identity to the sequence ofamino acids 22-185 of human Coco (SEQ ID NO:5), wherein said proteincomprises a modification with respect to the sequence of amino acids22-185 of SEQ ID NO: 5 such that cleavage in human serum is reduced oreliminated, and wherein said protein is substantially serum stable for aperiod of at least 24 hours.
 2. The myostatin antagonist protein ofclaim 1, wherein the protein comprises an amino acid sequence that hasat least 90% identity to the sequence of amino acids 22-189 of humanCoco.
 3. The myostatin antagonist protein of claim 1, wherein theprotein retains at least 50% of the myostatin antagonist activity afterexposure to human serum for 24 hours at 37° C.
 4. The myostatinantagonist protein of claim 1, wherein myostatin antagonist activity isassessed in an A204 cell based assay.
 5. The myostatin antagonistprotein of claim 1, wherein the protein comprises a modification withrespect to the amino acid sequence of SEQ ID NO:5 that reduces oreliminates cleavage within one or both of the following sequences ofhuman Coco: PARKRW (SEQ ID NO: 25) and SRRRVK (SEQ ID NO: 26).
 6. Apharmaceutical preparation comprising a myostatin antagonist protein ofclaim 1.